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Am J Physiol Endocrinol Metab 292: E1223-E1230, 2007. First published December 19, 2006; doi:10.1152/ajpendo.00446.2006
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Long-chain n-3 polyunsaturated fatty acids dissociate phosphorylation of Akt from phosphatidylinositol 3'-kinase activity in rats

Christelle Le Foll,1 Charlotte Corporeau,1 Valérie Le Guen,1 Jean-Paul Gouygou,2 Jean-Pascal Bergé,2 and Jacques Delarue3

1Equipe d'Accueil "Oxylipides", Faculté de Médecine, Brest; 2Département Génie Alimentaire, Ifremer, Nantes; and 3Equipe d'Accueil "Oxylipides" & Laboratoire Régional de Nutrition Humaine, Faculté de Médecine, Centre Hospitalo-Universitaire, Brest, France

Submitted 24 August 2006 ; accepted in final form 11 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined whether a low amount of dietary long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) modulated phosphatidylinositol 3'-kinase (PI 3-kinase) activity and downstream Akt phosphorylation differently in normal or insulin-resistant rats. Rats were fed for 28 days with either a control diet containing 14.6% of metabolizable energy (ME) as peanut-rape oil (PR) or an n-3 diet where 4.9% of ME as PR was replaced by fish oil. Over the last 5 days, rats received 9{per thousand} NaCl or dexamethasone (1 mg/kg). Insulin stimulation of both PI 3-kinase activity and Akt serine473 phosphorylation and modulation of GLUT4 content were studied in liver, muscle, and adipose tissue (AT). Glucose tolerance and insulin sensitivity were determined by an oral glucose challenge. In muscle and AT, LC n-3 PUFA abolished insulin-stimulated PI 3-kinase activity. These effects were not paralleled by defects in Akt serine473 phosphorylation, which was even increased in AT. Dexamethasone abolished insulin-stimulated PI 3-kinase activity in all tissues, whereas Akt serine473 phosphorylation was markedly reduced in muscle but unaltered in liver and AT. Such tissue-specific dissociating effects of LC n-3 PUFA on PI 3-kinase/Akt activation took place without alteration of glucose metabolism. Maintenance of a normal glucose metabolism by the n-3 diet despite abolition of PI 3-kinase activation was likely explained by a compensatory downstream Akt serine473 phosphorylation. The inability of LC n-3 PUFA to prevent insulin resistance by dexamethasone could result from the lack of such a dissociation.

eicosapentaenoic acid; docosahexaenoic acid; insulin resistance; dexamethasone

INSULIN RESISTANCE IS A COMMON CHARACTERISTIC of type 2 diabetes, obesity, and metabolic syndrome (36). Insulin resistance results from alterations of insulin signaling both in humans and in rodents. In skeletal muscle and in adipose tissue of patients with type 2 diabetes, phosphatidylinositol 3'-kinase (PI 3-kinase) activity is impaired (6, 30). In rats fed a high-fat diet rich in linoleic acid, insulin-induced PI 3-kinase activity is markedly depressed in liver and skeletal muscle, as well as its expression in adipose tissue (39). Moreover, in rats treated with a synthetic glucocorticoid (dexamethasone) that induces insulin resistance, PI 3-kinase activity stimulated by either insulin (32) or fed state (12) is deeply depressed in liver, muscle, and adipose tissue.

Downstream to PI 3-kinase, activation of serine-threonine kinase Akt, a protein that plays a pivotal role in regulation of glucose transporter 4 (GLUT4) trafficking (42), is impaired in both muscle and adipose tissue of patients with type 2 diabetes (43). In rats treated by dexamethasone, Akt serine473 phosphorylation is reduced by 50% in muscle (31), which may contribute to dexamethasone-induced impairment of glucose uptake in this tissue (31).

Consumption of long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), contained mainly in marine foodstuffs, is associated with a lower incidence of type 2 diabetes in Alaska and Greenland natives (1), ameliorates metabolic alterations associated to metabolic syndrome (including insulin resistance), prevents type 2 diabetes in Eskimos who gave up traditional marine foodstuffs (17, 18), increases insulin sensitivity (14), and partially prevents dexamethasone-induced insulin resistance in healthy humans (15). Substitution of one-third of sunflower oil (rich in linoleic acid) by fish oil into a high-fat diet (60% of energy as fat) in rats completely prevents the decrease of PI 3-kinase activity and expression in muscle and adipose tissue, respectively (39). Recently, we demonstrated that substitution of a low amount of fish oil, 4.9% of metabolizable energy (ME), into a normolipidic diet containing 14.6% of energy as peanut-rape oil induced, in fed rats, a decrease in PI 3-kinase activity in both liver and muscle while activating it in adipose tissue (12). The increase in PI 3-kinase activity in adipose tissue could have compensated for its decrease in liver and muscle, which could explain how glucose tolerance and insulin sensitivity remained unaffected (12). However, another explanation could be that Akt phosphorylation, downstream to PI 3-kinase, remained unaltered by fish oil, allowing insulin effects on glucose metabolism. In addition, in dexamethasone-treated rats, LC n-3 PUFA were unable to prevent either the decrease in PI 3-kinase activity in liver, muscle, and adipose tissue or whole body insulin resistance (12). This lack of preventive effect of LC n-3 PUFA could be explained by their inability to prevent, in addition to the decrease in PI 3-kinase activity, any concomitant dexamethasone-induced alteration of Akt phosphorylation.

Thus, the present study aimed to determine in fasted, insulin-injected rats 1) whether or not a low amount of LC n-3 PUFA could dissociate PI 3-kinase activation from Akt phosphorylation in liver, muscle, and adipose tissue and 2) whether or not such dissociation was abolished in dexamethasone-treated rats, which could explain the inability of LC n-3 PUFA to prevent insulin resistance.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care and tissue preparation. Forty-eight male Wistar rats (Elevage Janvier, Le Genest Saint Isle, France) aged 5 wk were housed in a temperature-, humidity-, and light-controlled room. For the first 5 wk preceding the study, rats were fed ad libitum with a standard chow diet (68% carbohydrates, 8% fat, 24% proteins; SNIFF R/S Elevage E diet, Elevage Janvier). Thirty-two rats were used for study of insulin signaling in liver, muscle, and adipose tissue (group 1), and 16 rats were used for study of glycemic and insulinemic responses to an oral glucose load (group 2).

For both groups, rats were randomly divided into two subgroups fed ad libitum for 4 wk with either a control diet (control) or a diet containing LC n-3 PUFA (n-3). Diets were prepared by Atelier de Préparation des Aliments Expérimentaux (APAE, INRA, Jouy-en-Josas, France). The control diet contained 14.6% of ME from fat as peanut-rape oil, 22% from casein, 42.4% from starch, and 21% from sucrose. The n-3 diet contained 9.7% of ME from fat as peanut-rape oil and 4.9% of ME from fat as fish oil, 22% from casein, 42.4% from starch, and 21% from sucrose. Five days before the end of the 4-wk period, rats in each of the four subgroups were randomly divided again into two subgroups to receive a daily intraperitoneal injection of either saline or dexamethasone (1 mg·kg–1·day–1) for 5 days to induce whole body insulin resistance (12) and alterations of PI 3-kinase activity in liver, muscle, and adipose tissue (12, 32). Food was withdrawn 12–14 h before animals were killed. On the last day, each of the four subgroups (n = 8) in group 1 (n = 32) was subdivided into two additional subgroups (n = 4), one receiving an intraperitoneal injection of 9% NaCl and the other receiving 100 U/kg of insulin (umuline rapide; Lilly France, Suresnes, France) 30 min before death. To minimize differences in time of being killed between treatments, eight rats were chosen randomly in the eight different subgroups and were killed simultaneously. All rats were killed in the morning, before 10 AM. Blood was immediately collected, and the plasma was separated by centrifugation and stored at –20°C until subsequent analysis. Liver, leg muscles (gastrocnemius, soleus, and biceps femoris), and epididymal fat tissue were immediately frozen, powdered into liquid nitrogen, and stored at –80°C. The protocol was performed in conformity with regulations for the care and use of laboratory animals (decree no. 87-848 and agreement no. 29030 from the French Department of Agriculture and Fishing) and the American Physiological Society's Guiding Principles in the Care and Use of Animals.

Oral glucose tolerance test. An oral glucose tolerance test (OGTT) (3 g/kg body wt) was performed on rats in group 2 after 12–14 h of fasting. A local anesthetization with lidocaine (5% EMLA cream; AstraZeneca, Rueil-Malmaison, France) was performed on the tail; a vein blood sample was taken first, and then a glucose solution was orally administrated. Blood samples were collected at 10, 20, 30, 45, 60, 90, and 120 min. For each sample, a fraction was immediately used for glucose determination with a GLUCOTOUCH Pro Glucometer (Lifescan, Milpitas, CA), another fraction was collected in tubes containing heparin, and plasma was separated by centrifugation and stored at –20°C until insulin concentration determination.

Plasma glucose, insulin, and nonesterified fatty acid concentrations. Except for OGTT, glycemia was determined with the glucose oxidase method by using an automated multiparametric analyzer (Modular; Roche, Mannheim, Germany). Plasma insulin levels were determined by a radioimmunoassay kit (Linco Research, St. Charles, MO), using rat insulin as standard. Plasma nonesterified fatty acid (NEFA) concentration was determined by a colorimetric method using a NEFA C kit (Wako Chemical, Neuss, Germany).

Extraction and analysis of lipids in diets and tissues. Total lipid extracts from diets and rat tissues were obtained by the method of Bligh and Dyer (9). For rat tissues, neutral lipids, galactolipids, and phospholipids were successively eluted by CHCl3 (1 ml/mg crude lipids), Me2CO-MeOH (9:1; 1.5 ml/mg crude lipids), and MeOH (1 ml/mg crude lipids), as previously described by Bergé et al. (7). Lipids were evaporated under nitrogen and transmethylated by contact with MeOH-H2SO4 (98:2) in excess for one night at 50°C. After cooling, 2 ml of pentane and 1 ml of water were added and vortexed. The upper organic phases containing fatty acid methyl esters were collected and assayed by gas chromatography using a PerkinElmer Autosystem equipped with an flame ionization detector. Separation was done using helium as carrier gas on a fused silica column (BPX, 70.60 m long, 0.25 mm ID, 0.25 µm film thickness; SGE Analytical Science, Courtaboeuf, France) programmed from 55 (for 2 min) to 150°C at 20°C/min. Fatty acid methyl esters were identified by comparison of their equivalent chain lengths with those of authentic standards, and quantification was done using margaric acid (17:0) as internal standard (8, 26a).

Western blot analysis. Powdered tissues (1 g) were solubilized in ice-cold buffer containing 150 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% Igepal, 2 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 100 mM NaF, 10 nM Na4P2O7, and 2 mM Na3VO4, pH 7.4. Homogenates were centrifuged at 10,000 g for 1 h at 4°C. Protein concentration in supernatants was determined by a DC protein assay (Bio-Rad, Hercules, CA). Lysates were separated by SDS-PAGE, and immunoblotting was performed using antibodies directed against either p85{alpha} subunit of PI 3-kinase (Santa Cruz Biotechnology, Santa Cruz, CA), Akt and phosphoserine473 Akt (Cell Signaling Technology, Danvers, MA), or GLUT4 (Chemicon International, Temecula, CA). Blots were revealed by enhanced chemiluminescence anti-rabbit horseradish peroxidase detection kit (Amersham Bioscience, Buckinghamshire, UK). Band intensities were quantified by optical densitometry (Gel Doc XR scanning software; Bio-Rad).

PI 3-kinase activity. Powdered tissues (1 g) were solubilized in ice-cold buffer containing 137 mM NaCl, 20 mM Tris, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Igepal, 2 mM PMSF, 10 µg/ml aprotinin, and 0.15 mM Na3VO4, pH 7.5. Immunoprecipitation was performed using anti-insulin receptor substrate-1 antibody (Upstate Biotechnology, Lake Placid, NY). After being washed, the immune complex was used to measure PI 3-kinase, as previously described (12). Phosphorylated PI was extracted and resolved onto silica gel TLC plate (Whatman, Middlesex, UK). TLC plates were developed in CHCl3-CH3OH-H2O-NH4OH (120:94:22.6:4), dried, and visualized by autoradiography. The radioactivity in spots that comigrated with a PI-4 standard was measured by Instant Imager (Packard, Palo Alto, CA).

Statistics. Data are presented as means ± SE. Statistical analyses were performed by using a Mann-Whitney test. Software was Statview II (Abacus Concepts), running on a Powerbook G4 (Apple, Cupertino, CA). A value of P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Characteristics of experimental rats. The n-3 diet had no effect on body weight, food intake, glycemia, insulinemia, or plasma NEFA concentration (Table 1). Dexamethasone treatment induced a decrease in weight and food intake (P < 0.05; Table 1) as well as a marked increase in glycemia, insulinemia, and plasma NEFA concentration (P < 0.05). The n-3 diet did not alter these effects of dexamethasone (Table 1). In all groups of rats, the intraperitoneal insulin injections raised insulinemia to similar levels at the time tissues were removed (Table 1).


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  		Table 1. Animal characteristics during the 5-day period of dexa treatment

 
Effect of n-3 diet on glycemic and insulinemic responses to OGTT. The n-3 diet had no effect on mean glycemic (control = 7.09 ± 0.49 mM; n-3 = 7.23 ± 0.27 mM) and insulinemic (control = 0.16 ± 0.06 nM; n-3 = 0.18 ± 0.04 nM) responses to oral glucose. As expected, dexamethasone treatment induced a higher mean glycemic (control = 7.09 ± 0.49 mM vs. control + Dexa = 15.05 ± 2.82 mM, P < 0.05) and insulinemic (control = 0.16 ± 0.06 nM vs. control + Dexa = 2.17 ± 0.55 nM, P < 0.05) responses to oral glucose. The n-3 diet did not alter these effects of dexamethasone (mean of glycemia: n-3 + Dexa = 18.41 ± 4.63 mM; mean of insulinemia: n-3 + Dexa = 1.97 ± 0.41 ng/ml).

Effect of n-3 diet on fatty acid content of membrane phospholipids in tissues. Fatty acid content of membrane phospholipids of liver, muscle, and adipose tissue is shown in Fig. 1. The proportion of LC n-3 PUFA into phospholipids of the three tissues was higher in rats fed the n-3 diet than in control rats, demonstrating their incorporation into membranes. The incorporation of n-3 LC-PUFA into adipose tissue and in muscle phospholipids was 8- and 3.5-fold greater with n-3 diet than with control diet, whereas it was only 1.6 times greater in liver.


Figure 1
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  		Fig. 1. Fatty acid composition of membrane phospholipids of liver, muscle, and adipose tissue (AT) expressed as n-3 polyunsaturated fatty acids (PUFA)/total fatty acids (TFA). Data are means ± SE; n = 8 rats/experiment. *P < 0.05 vs. control; {dagger}P < 0.01 vs. control. dexa, Dexamethasone.

 
Effect of n-3 diet on PI 3-kinase activity and Akt phosphorylation in liver. In liver, p85{alpha} subunit of PI 3-kinase and Akt protein contents were unchanged, whatever the diet, in control and dexamethasone-treated rats, as determined by densitometric quantification on blots (data not shown). The n-3 diet tended to decrease the ability of insulin to stimulate PI 3-kinase activity (Fig. 2A) and did not modulate insulin-induced Akt serine473 phosphorylation (Fig. 2B). Dexamethasone alone depressed the ability of insulin to stimulate PI 3-kinase activity (P < 0.05; Fig. 2A), whereas it did not alter Akt serine473 phosphorylation (Fig. 2B). Moreover, the n-3 diet did not modify the dexamethasone-induced inhibition of PI-3 kinase activity (Fig. 2A), but it abolished insulin-induced Akt serine473 phosphorylation (P < 0.05; Fig. 2B).


Figure 2
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  		Fig. 2. Phosphatidylinositol 3'-kinase (PI 3-kinase) activity (A) and Akt serine473 (Ser473) phosphorylation (B) in liver. Blots are representative of 4 experiments. Results are expressed as means of insulin/placebo ± SE; n = 4 rats. *P < 0.05 vs. control.

 
Effect of n-3 diet on PI 3-kinase activity, Akt phosphoylation, and GLUT4 content in muscle. In muscle, p85{alpha} subunit of PI 3-kinase and Akt protein contents were unchanged, whatever the diet, in control and dexamethasone-treated rats, as determined by densitometric quantification on blots (data not shown). In contrast to liver, the n-3 diet abolished the ability of insulin to stimulate PI 3-kinase activity (P < 0.05; Fig. 3A) and tended to diminished the level of insulin-induced Akt serine473 phosphorylation (Fig. 3B). Dexamethasone alone abolished the ability of insulin to stimulate PI 3-kinase activity (P < 0.05; Fig. 3A), whereas it allowed insulin-induced Akt serine473 phosphorylation at a lower level than in control rats (P < 0.05; Fig. 3B). The n-3 diet did not alter these effects of dexamethasone on either PI 3-kinase activation or Akt serine473 phosphorylation. Moreover, GLUT4 expression, as determined by densitometric quantification on blots, remained unchanged, whatever the diet and treatment (Fig. 5A).


Figure 3
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  		Fig. 3. PI 3-kinase activity (A) and Akt Ser473 phosphorylation (B) in muscle. Blots are representative of 4 experiments. Results are expressed as means of insulin/placebo ± SE; n = 4 rats. *P < 0.05 vs. control.

 

Figure 5
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  		Fig. 5. Glucose transporter 4 (GLUT4) protein content in muscle (A) and AT (B). Blots are representative of 4 experiments; n = 4 rats. Data are means ± SE.

 
Effect of n-3 diet on PI 3-kinase activity, Akt phosphorylation, and GLUT4 content in adipose tissue. In adipose tissue, p85{alpha} subunit of PI 3-kinase and Akt protein contents were unchanged, whatever the diet, in control and dexamethasone-treated rats, as determined by densitometric quantification on blots (data not shown). As in muscle, the n-3 diet abolished the ability of insulin to stimulate PI 3-kinase activity (P < 0.05; Fig. 4A). In contrast, the n-3 diet increased the ability of insulin to stimulate Akt serine473 phosphorylation by 113% (P < 0.05; Fig. 4B) compared with control rats. Dexamethasone alone abolished the ability of insulin to stimulate PI 3-kinase activity (P < 0.05; Fig. 4A), whereas it did not alter Akt serine473 phosphorylation (Fig. 4B). The n-3 diet did not modulate these effects of dexamethasone. Moreover, GLUT4 expression, as determined by quantification on blots, remained unchanged, whatever the diet and treatment (Fig. 5B).


Figure 4
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  		Fig. 4. PI 3-kinase activity (A) and Akt Ser473 phosphorylation (B) in adipose tissue. Blots are representative of 4 experiments. Results are expressed as means of insulin/placebo ± SE; n = 4 rats. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our study clearly demonstrates that a low amount of LC n-3 PUFA [eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) = 0.93% of energy intake] given as dietary fish oil modulates insulin-stimulated Akt serine473 phosphorylation independently of insulin-stimulated PI 3-kinase activity in normal and insulin-resistant rats. This dissociation could explain, at least partially, the discrepancy between alteration of PI 3-kinase activation by LC n-3 PUFA in muscle and adipose tissue and the maintenance of whole body insulin sensitivity in normal rats.

In rats fed with the control diet and not treated by dexamethasone, insulin stimulated, as expected, both PI 3-kinase activity and Akt serine473 phosphorylation in liver, muscle, and adipose tissue. In contrast, n-3 diet abolished insulin-induced activation of PI 3-kinase in muscle and adipose tissue, but not in liver. This negative effect of n-3 diet on PI 3-kinase activation was dissociated from that on Akt serine473 phosphorylation. Indeed, the stimulation of Akt phosphorylation by insulin was reduced 40% in muscle; this reduction was not significant. However, although this reduction has been significant, Akt remained highly activated (4-fold) by insulin. In adipose tissue, Akt phosphorylation was about 100% increased. Thus, LC n-3 PUFA had both a tissue-specific effect on PI 3-kinase activity, as previously reported (12, 39), and a tissue-specific dissociating effect on stimulation of PI 3-kinase activity and Akt phosphorylation. This observation is new. Some studies have reported a normal stimulation of Akt phosphorylation contrasting with an inhibition of PI 3-kinase activation in insulin-resistant cultured cells, animals, or humans. Indeed, in human insulin-resistant muscle, PI 3-kinase activity was decreased twofold, whereas Akt serine473 phosphorylation was similar to normal subjects (21, 38). Likewise, a lack of PI 3-kinase activation contrasting with a stimulation of Akt phosphorylation has been reported in adipocytes of male insulin-resistant BtB6 mice (28) or in adenovirus-transformed 3T3-L1 adipocytes (41). Other studies performed in cultured cells observed either an inhibiting effect of LC n-3 PUFA on Akt phosphorylation or its stimulation. In mouse neuroblastoma cells (neuronal 2A), the addition of DHA in the culture medium did not alter insulin-induced PI 3-kinase activity but facilitated translocation and phosphorylation of Akt (2). In a murine monocytic cell line (23), DHA inhibited the phosphorylation of Akt induced by lipopolysaccharide or by lauric acid (a saturated fatty acid), probably by inhibiting PI 3-kinase activity. In hepatoma cells, EPA increased both PI 3-kinase activity, an effect additive to this of insulin, and Akt phosphorylation (27). The basic mechanisms sustaining the tissue-specific effects of LC n-3 PUFA in our study remain unclear. The abolition of PI 3-kinase activity in muscle and adipose tissue and the increase in Akt serine473 phosphorylation in adipose tissue were not explained by any modulation of their protein contents by the n-3 diet. However, an alteration of membranes lipid composition could have contributed to these effects of LC n-3 PUFA. In the present study, the proportion of LC n-3 PUFA in membranes phospholipids increased in liver, muscle, and adipose tissue. The mechanisms sustaining the decreasing effect of dexamethasone on LC n-3 PUFA content in liver phospholipids remain unclear. Dexamethasone has not been reported to increase phospholipases activity in liver. Although not specifically determined in our study, LC n-3 PUFA was probably incorporated not only into plasma membranes, including lipid rafts and caveolin microdomains, but also into membranes of endosomes and intracellular vesicles. LC n-3 PUFA were shown to alter the lipid microdomains of plasma membrane and to change microlocalization of different signaling proteins (10, 19, 24–26). Because PI 3-kinase and Akt are compartmentalized and activated through their translocation to plasma membrane (4, 5, 16), an alteration of membrane fatty acids content could alter their activation. Many data sustain this hypothesis. In rat smooth muscle cells, LC n-3 PUFA increased intracellular caveolin-1, which is known to regulate PI 3-kinase/Akt signaling pathway (34). In neuronal 2A cells cultured in a medium enriched with DHA, the increase in Akt phosphorylation was due to an increase in its translocation, with this effect itself being due to the ability of DHA to increase phosphatidylserine in cell membranes (2). In breast cancer cells with high Akt activity, EPA decreased Akt phosphorylation by interfering with its translocation to plasma membranes (13). Because the translocation of Akt to the plasma membrane requires its interaction with acidic phospholipids in membrane (40), an alteration of membrane composition can alter Akt translocation. Another possibility to explain the effect of n-3 diet is the unsaturation of phosphatidylinositide (3, 4, 5) P3 (PIP3) produced by PI 3-kinase. PIP3 generated in the inner leaflet of the plasma membrane is converted to PIP2 by a specific phosphatase. PIP3 and PIP2 activate phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates Akt. PIP3 derivatives containing unsaturated fatty acids are much more potent activators of PDK1 than derivatives containing saturated fatty acids (3). Thus, an unsaturation of PIP3 resulting from the n-3 diet could have increased PDK1 activity and thus Akt phosphorylation in adipose tissue of rats fed the n-3 diet. Recently, the mammalian PI3K/PTEN/Akt pathway was reconstituted in yeast Saccharomyces cerevisiae (29). This model offers the opportunity to study whether or not LC n-3 PUFA could alter PIP2 and PIP3 generation, as well as PDK1 activity. This is currently under investigation. Last, the dissociation effect by LC n-3 PUFA on PI 3-kinase and Akt activation may involve another kinase capable of activating Akt in response to insulin through a PI 3-kinase-independent pathway. The new complex mTOR-rictor-GbetaL could play a role in this process, since Sarbassov et al. (33) recently demonstrated its implication in the regulation of Akt serine473 phosphorylation. However, the possible modulation of this pathway by LC n-3 PUFA has not been studied in the present study.

In dexamethasone-treated rats fed with the control diet, PI 3-kinase activation by insulin was markedly decreased or abolished in liver, muscle, and adipose tissue, as previously reported (12, 32). Akt serine473 phosphorylation was deeply reduced in muscle but remained unaltered in liver and adipose tissue. These effects of dexamethasone were observed in the absence of any modulation of PI 3-kinase and Akt protein contents. Previous studies (31) have shown that Akt phosphorylation was depressed in muscle of dexamethasone-treated rats. In isolated rat adipocytes, dexamethasone induced a 40% decrease in insulin-activated Akt serine473 phosphorylation (11), but PI 3-kinase activity was not determined. As previously reported (12), the n-3 diet did not modify the dexamethasone-induced inhibition of PI 3-kinase activation in the three tissues. The n-3 diet also did not modify the effects of dexamethasone on Akt serine473 phosphorylation in muscle and adipose tissue but abolished it in liver, also translating a tissue-specific dissociating effect.

The dissociating effect of LC n-3 PUFA on insulin-stimulated PI 3-kinase activity and Akt phosphorylation brings a new highlight to the maintenance in normal rats of whole body glucose tolerance and insulin sensitivity despite the abolition of PI 3-kinase activity in muscle and liver. Insulin signaling transduction on glucose metabolism is likely to have been maintained because the abolition of PI 3-kinase activation was bypassed by maintenance (muscle) and by increase (adipose tissue) of Akt activation, allowing downstream stimulation of GLUT4 translocation. Indeed, Venable et al. (41) reported unchanged glucose uptake in adipocytes with inhibition of PI 3-kinase activity and normal Akt serine473 phosphorylation, concluding with a novel PI 3-kinase-independent pathway involved in the regulation of GLUT4 translocation. Taken together, these data and ours (22, 37, 41) suggest that activation of PI 3-kinase may be neither necessary nor sufficient for transduction of the effects of insulin on glucose metabolism. In dexamethasone-treated rats fed with the control diet, insulin resistance and glucose intolerance occurred, as demonstrated by the higher insulinemic and glycemic responses to oral glucose, because both PI 3-kinase and Akt activation were deeply altered in muscle, the main tissue where glucose is taken up during an oral glucose load. Last, as previously reported, the n-3 diet was unable to prevent dexamethasone-induced alteration of glucose metabolism (12) since LC n-3 PUFA did not prevent the decrease of Akt phosphorylation in muscle and decreased it in adipose tissue, although it was completely abolished in liver. The inability of LC n-3 PUFA to prevent dexamethasone alteration could be due to a too potent effect of the high dose of dexamethasone. It cannot be excluded that a lower dose of dexamethasone would have less deleterious effects on insulin signaling, allowing LC n-3 PUFA to prevent dexamethasone-induced alterations. We deliberately chose, in the present study, this high dose of dexamethasone, as previously used (12), because our hypothesis was that a possible mechanism of the lack of preventive effect of LC n-3 PUFA toward insulin resistance could be explained by its inability to dissociate alteration of PI-3 kinase activity from that of Akt. It would be interesting to carry out a study using a lower dose of dexamethasone, but one sufficient to induce insulin resistance. To our knowledge, the lowest dose of dexamethasone used in rats to induce insulin resistance was 5 µg·kg–1·day–1 over 1 wk (35). Thus, an additional study initially using this low dose could be performed to determine whether LC n-3 PUFA could prevent or not prevent both insulin resistance and alterations of insulin signaling. In conclusion, LC n-3 PUFA dissociate insulin-induced activation of PI 3-kinase activity from Akt phosphorylation in a tissue-specific manner. This dissociating effect can explain the absence of alteration of glucose tolerance and insulin sensitivity despite the abolition of PI 3-kinase activation in muscle and adipose tissue by LC n-3 PUFA. Thus, Akt and intermediates between PI 3-kinase and Akt, such as PIP2, PIP3, and PDK1, could be considered specific targets of LC n-3 PUFA. The inability of LC n-3 PUFA to prevent dexamethasone-induced glucose intolerance and insulin resistance could result from the inability of LC n-3 PUFA in muscle.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 GRANTS
REFERENCES
 
This work was supported by a grant from Région Bretagne and Région Pays de la Loire.


   ACKNOWLEDGMENTS
 
We thank Danièle Lucas and Marie-Pierre Moineau for the assays of glucose and insulin and Nicole Hourmant for technical help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Delarue, Laboratoire Régional de Nutrition Humaine, CHU Cavale Blanche F-29200-Brest, France (e-mail: jacques.delarue{at}univ-brest.fr)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adler AI, Boyko EJ, Schraer CD, Murphy NJ. Lower prevalence of impaired glucose tolerance and diabetes associated with daily seal oil or salmon consumption among Alaska Natives. Diabetes Care 17: 1498–1501, 1994.[Abstract]
  2. Akbar M, Calderon F, Wen Z, Kim HY. Docosahexaenoic acid: a positive modulator of Akt signaling in neuronal survival. Proc Natl Acad Sci USA 102: 10858–10863, 2005.[Abstract/Free Full Text]
  3. Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PR, Reese CB, Cohen P. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7: 261–269, 1997.[CrossRef][ISI][Medline]
  4. Andjelkovic M, Jakubowicz T, Cron P, Ming XF, Han JW, Hemmings BA. Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors. Proc Natl Acad Sci USA 93: 5699–5704, 1996.[Abstract/Free Full Text]
  5. Balbis A, Baquiran G, Bergeron JJ, Posner BI. Compartmentalization and insulin-induced translocations of insulin receptor substrates, phosphatidylinositol 3-kinase, and protein kinase B in rat liver. Endocrinology 141: 4041–4049, 2000.[Abstract/Free Full Text]
  6. Bandyopadhyay GK, Yu JG, Ofrecio J, Olefsky JM. Increased p85/55/50 expression and decreased phosphotidylinositol 3-kinase activity in insulin-resistant human skeletal muscle. Diabetes 54: 2351–2359, 2005.[Abstract/Free Full Text]
  7. Bergé JP, Gouygou JP, Dubacq JP, Durand P. Reassessment of lipid composition of the diatom, Skeletonema costatum. Phytochemistry 39: 1017–1021, 1995.[CrossRef][ISI]
  8. Berge JP, Debiton E, Dumay J, Durand P, Barthomeuf C. In vitro anti-inflammatory and anti-proliferative activity of sulfolipids from the red alga Porphyridium cruentum. J Agric Food Chem 50: 6227–6232, 2002.[CrossRef][ISI][Medline]
  9. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917, 1959.[Medline]
  10. Bousserouel S, Raymondjean M, Brouillet A, Bereziat G, Andreani M. Modulation of cyclin D1 and early growth response factor-1 gene expression in interleukin-1beta-treated rat smooth muscle cells by n-6 and n-3 polyunsaturated fatty acids. Eur J Biochem 271: 4462–4473, 2004.[ISI][Medline]
  11. Buren J, Liu HX, Jensen J, Eriksson JW. Dexamethasone impairs insulin signalling and glucose transport by depletion of insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase B in primary cultured rat adipocytes. Eur J Endocrinol 146: 419–429, 2002.[Abstract]
  12. Corporeau C, Le Foll C, Taouis M, Gouygou JP, Berge JP, Delarue J. Adipose tissue compensates for defect of phosphatidylinositol 3'-kinase induced in liver and muscle by dietary fish oil in fed rats. Am J Physiol Endocrinol Metab 290: E78–E86, 2006.[Abstract/Free Full Text]
  13. DeGraffenried LA, Friedrichs WE, Fulcher L, Fernandes G, Silva JM, Peralba JM, Hidalgo M. Eicosapentaenoic acid restores tamoxifen sensitivity in breast cancer cells with high Akt activity. Ann Oncol 14: 1051–1056, 2003.[Abstract/Free Full Text]
  14. Delarue J, Couet C, Cohen R, Brechot JF, Antoine JM, Lamisse F. Effects of fish oil on metabolic responses to oral fructose and glucose loads in healthy humans. Am J Physiol Endocrinol Metab 270: E353–E362, 1996.[Abstract/Free Full Text]
  15. Delarue J, Li CH, Cohen R, Corporeau C, Simon B. Interaction of fish oil and a glucocorticoid on metabolic responses to an oral glucose load in healthy humans. Br J Nutr 95: 267–272, 2006.[CrossRef][ISI][Medline]
  16. Drake PG, Balbis A, Wu J, Bergeron JJ, Posner BI. Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver. Am J Physiol Endocrinol Metab 279: E266–E274, 2000.[Abstract/Free Full Text]
  17. Ebbesson SO, Ebbesson LO, Swenson M, Kennish JM, Robbins DC. A successful diabetes prevention study in Eskimos: the Alaska Siberia project. Int J Circumpolar Health 64: 409–424, 2005.[Medline]
  18. Ebbesson SO, Risica PM, Ebbesson LO, Kennish JM, Tejero ME. Omega-3 fatty acids improve glucose tolerance and components of the metabolic syndrome in Alaskan Eskimos: the Alaska Siberia project. Int J Circumpolar Health 64: 396–408, 2005.[Medline]
  19. Fan YY, McMurray DN, Ly LH, Chapkin RS. Dietary (n-3) polyunsaturated fatty acids remodel mouse T-cell lipid rafts. J Nutr 133: 1913–1920, 2003.[Abstract/Free Full Text]
  20. Kim YB, Nikoulina SE, Ciaraldi TP, Henry RR, Kahn BB. Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes. J Clin Invest 104: 733–741, 1999.[ISI][Medline]
  21. Krook A, Moller DE, Dib K, O'Rahilly S. Two naturally occurring mutant insulin receptors phosphorylate insulin receptor substrate-1 (IRS-1) but fail to mediate the biological effects of insulin. Evidence that IRS-1 phosphorylation is not sufficient for normal insulin action. J Biol Chem 271: 7134–7140, 1996.[Abstract/Free Full Text]
  22. Lee JY, Ye J, Gao Z, Youn HS, Lee WH, Zhao L, Sizemore N, Hwang DH. Reciprocal modulation of Toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids. J Biol Chem 278: 37041–37051, 2003.[Abstract/Free Full Text]
  23. Li Q, Ma J, Tan L, Wang C, Li N, Li Y, Xu G, Li J. Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts. Sci China C Life Sci 49: 63–72, 2006.[CrossRef][ISI][Medline]
  24. Li Q, Tan L, Wang C, Li N, Li Y, Xu G, Li J. Polyunsaturated eicosapentaenoic acid changes lipid composition in lipid rafts. Eur J Nutr 45: 144–151, 2006.[CrossRef][ISI][Medline]
  25. Ma DW, Seo J, Davidson LA, Callaway ES, Fan YY, Lupton JR, Chapkin RS. n-3 PUFA alter caveolae lipid composition and resident protein localization in mouse colon. FASEB J 18: 1040–1042, 2004.[Abstract/Free Full Text]
  26. Mol HGJ, Janssen HG, Cramers CA. Large volume sample introduction using temperature programmable injectors: implications of liner diameter. J High Res Chromatogr 18: 19–27, 1995.[CrossRef]
  27. Murata M, Kaji H, Iida K, Okimura Y, Chihara K. Dual action of eicosapentaenoic acid in hepatoma cells: up-regulation of metabolic action of insulin and inhibition of cell proliferation. J Biol Chem 276: 31422–31428, 2001.[Abstract/Free Full Text]
  28. Nadler ST, Stoehr JP, Rabaglia ME, Schueler KL, Birnbaum MJ, Attie AD. Normal Akt/PKB with reduced PI3K activation in insulin-resistant mice. Am J Physiol Endocrinol Metab 281: E1249–E1254, 2001.[Abstract/Free Full Text]
  29. Rodriguez-Escudero I, Roelants FM, Thorner J, Nombela C, Molina M, Cid VJ. Reconstitution of the mammalian PI3K/PTEN/Akt pathway in yeast. Biochem J 390: 613–623, 2005.[CrossRef][ISI][Medline]
  30. Rondinone CM, Carvalho E, Wesslau C, Smith UP. Impaired glucose transport and protein kinase B activation by insulin, but not okadaic acid, in adipocytes from subjects with Type II diabetes mellitus. Diabetologia 42: 819–825, 1999.[CrossRef][ISI][Medline]
  31. Ruzzin J, Wagman AS, Jensen J. Glucocorticoid-induced insulin resistance in skeletal muscles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3 inhibitor. Diabetologia 48: 2119–2130, 2005.[CrossRef][ISI][Medline]
  32. Saad MJ, Folli F, Kahn JA, Kahn CR. Modulation of insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of dexamethasone-treated rats. J Clin Invest 92: 2065–2072, 1993.[ISI][Medline]
  33. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098–1101, 2005.[Abstract/Free Full Text]
  34. Sedding DG, Hermsen J, Seay U, Eickelberg O, Kummer W, Schwencke C, Strasser RH, Tillmanns H, Braun-Dullaeus RC. Caveolin-1 facilitates mechanosensitive protein kinase B (Akt) signaling in vitro and in vivo. Circ Res 96: 635–642, 2005.[Abstract/Free Full Text]
  35. Severino C, Brizzi P, Solinas A, Secchi G, Maioli M, Tonolo G. Low-dose dexamethasone in the rat: a model to study insulin resistance. Am J Physiol Endocrinol Metab 283: E367–E373, 2002.[Abstract/Free Full Text]
  36. Shulman GI. Cellular mechanisms of insulin resistance. J Clin Invest 106: 171–176, 2000.[ISI][Medline]
  37. Staubs PA, Nelson JG, Reichart DR, Olefsky JM. Platelet-derived growth factor inhibits insulin stimulation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase in 3T3-L1 adipocytes without affecting glucose transport. J Biol Chem 273: 25139–25147, 1998.[Abstract/Free Full Text]
  38. Storgaard H, Song XM, Jensen CB, Madsbad S, Bjornholm M, Vaag A, Zierath JR. Insulin signal transduction in skeletal muscle from glucose-intolerant relatives of type 2 diabetic patients [corrected]. Diabetes 50: 2770–2778, 2001.[Abstract/Free Full Text]
  39. Taouis M, Dagou C, Ster C, Durand G, Pinault M, Delarue J. N-3 polyunsaturated fatty acids prevent the defect of insulin receptor signaling in muscle. Am J Physiol Endocrinol Metab 282: E664–E671, 2002.[Abstract/Free Full Text]
  40. Thomas CC, Deak M, Alessi DR, van Aalten DM. High-resolution structure of the pleckstrin homology domain of protein kinase b/akt bound to phosphatidylinositol (3,4,5)-trisphosphate. Curr Biol 12: 1256–1262, 2002.[CrossRef][ISI][Medline]
  41. Venable CL, Frevert EU, Kim YB, Fischer BM, Kamatkar S, Neel BG, Kahn BB. Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/Protein kinase B activation. J Biol Chem 275: 18318–18326, 2000.[Abstract/Free Full Text]
  42. Welsh GI, Hers I, Berwick DC, Dell G, Wherlock M, Birkin R, Leney S, Tavare JM. Role of protein kinase B in insulin-regulated glucose uptake. Biochem Soc Trans 33: 346–349, 2005.[CrossRef][ISI][Medline]
  43. Zdychova J, Komers R. Emerging role of Akt kinase/protein kinase B signaling in pathophysiology of diabetes and its complications. Physiol Res 54: 1–16, 2005.[ISI][Medline]




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