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J Appl Physiol 98: 1037-1043, 2005. First published November 5, 2004; doi:10.1152/japplphysiol.00536.2004
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Endurance exercise training increases insulin responsiveness in isolated adipocytes through IRS/PI3-kinase/Akt pathway

Sidney B. Peres,1 Solange M. Franzói de Moraes,2 Cecilia E. M. Costa,2 Luciana C. Brito,1 Julie Takada,1 Sandra Andreotti,1 Magaly A. Machado,1 Maria Isabel C. Alonso-Vale,1 Cristina N. Borges-Silva,1 and Fabio B. Lima1

1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo; and 2Department of Morphophysiological Sciences, University of Maringa, Parana, Brazil

Submitted 20 May 2004 ; accepted in final form 3 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endurance exercise training promotes important metabolic adaptations, and the adipose tissue is particularly affected. The aim of this study was to investigate how endurance exercise training modulates some aspects of insulin action in isolated adipocytes and in intact adipose tissue. Male Wistar rats were submitted to daily treadmill running (1 h/day) for 7 wk. Sedentary age-matched rats were used as controls. Final body weight, body weight gain, and epididymal fat pad weight did not show any statistical differences between groups. Adipocytes from trained rats were smaller than those from sedentary rats (205 ± 16.8 vs. 286 ± 26.4 pl; P < 0.05). Trained rats showed decreased plasma glucose (4.9 ± 0.13 vs. 5.3 ± 0.07 mM; P < 0.05) and insulin levels (0.24 ± 0.012 vs. 0.41 ± 0.049 mM; P < 0.05) and increased insulin-stimulated glucose uptake (23.1 ± 3.1 vs. 12.1 ± 2.9 pmol/cm2; P < 0.05) compared with sedentary rats. The number of insulin receptors and the insulin-induced tyrosine phosphorylation of insulin receptor-{beta} subunit did not change between groups. Insulin-induced tyrosine phosphorylation insulin receptor substrates (IRS)-1 and -2 increased significantly (1.57- and 2.38-fold, respectively) in trained rats. Insulin-induced IRS-1/phosphatidylinositol 3 (PI3)-kinase (but not IRS-2/PI3-kinase) association and serine Akt phosphorylation also increased (2.06- and 3.15-fold, respectively) after training. The protein content of insulin receptor-{beta} subunit, IRS-1 and -2, did not differ between groups. Taken together, these data support the hypothesis that the increased adipocyte responsiveness to insulin observed after endurance exercise training is modulated by IRS/PI3-kinase/Akt pathway.

adipose tissue; insulin signaling; exercise; diabetes; obesity

ENDURANCE EXERCISE TRAINING has been recognized as an important therapeutic tool used to treat and prevent obesity and Type 2 diabetes mellitus, diseases that are characterized by an increased insulin resistance leading to a diminished peripheral glucose uptake (3, 4). The adhesion to a program of physical exercise training can improve many metabolic and hormonal functions in different tissues. Muscle and adipose tissue become more responsive to insulin, and a reduction in fat depots (both visceral and subcutaneous) has been consistently reported (5, 11, 13, 14, 28). The increase in insulin-induced glucose uptake rates reported after chronic exercise is partially due to the rise in the activity and expression of the proteins involved in insulin signaling as well as in the insulin responsive glucose transporter (GLUT4), and this response is particularly known in skeletal muscle (11, 17, 21, 28). However, the effects of endurance exercise training, mainly its influence on the early steps of the insulin signaling pathway in intact adipose tissue or isolated adipocytes, have not been adequately evaluated. In the present work, we analyzed the protein content and the phosphorylation degree of insulin receptor-{beta} subunit (IR-{beta}), insulin receptor substrates (IRS)-1 and -2, and the association of IRS proteins with phosphatidylinositol-3-kinase (PI3-K). We also determined the ability of insulin to induce serine phosphorylation of Akt/PKB in endurance exercise-trained rats. Our results show that endurance exercise training improved the responsiveness of adipocytes to insulin and that this effect is exerted through the IRS/PI3-K/Akt pathway.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and training schedule.   Male Wistar rats (45 days old, weight 200 g) from the Animal Resources Center of the Institute of Biomedical Sciences of the University of São Paulo were used in the experiments. They were housed in collective cages with free access to water and food (balanced chow pellet diet, Nuvilab CR1, Nuvital SA, Colombo, Brazil). The animals were randomly distributed into two age-matched groups: sedentary or exercise-trained. The last group was submitted to an endurance exercise training program consisting of running sessions on a motor treadmill (KT-300, Inbramed, Porto Alegre, Brazil) for 1 h/day, 5 days/wk for 7 wk. The training schedule was based on Braga et al. (9), with some adaptations. Briefly, the exercise was progressive with increasing intensity, determined by a combination of velocity and duration (0% grade). It began with 5 m/min, 10–15 min/day and reached 8.3 m/min and 30 min/day at the end of week 1. The training intensity was gradually increased up to 60 min/day (by week 3) with a speed of 16.7 m/min (by weeks 4–5) and finally 20 m/min (by weeks 6–7). Under these conditions, the training allowed ~60% of maximal oxygen consumption for the last 2 wk. The protocol used for oxygen consumption testing was performed as described elsewhere (9). The Ethical Committee for Animal Research of the Institute of Biomedical Sciences of the University of Sao Paulo approved all experimental procedures.

Materials.   Antiphosphotyrosine, anti-IR-{beta}, anti-IRS-1, and anti-IRS-2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-p85 regulatory subunit of PI3-K antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-phosphoserine473-Akt antibody was from Cell Signaling Technology (Beverly, MA). Reagents for SDS polyacrylamide gel electrophoresis and immunoblotting were from Bio-Rad (Richmond, CA). Collagenase type II, aprotinin, dithiothreitol, HEPES, phenylmethylsulfonyl fluoride, sodium orthovanadate, Triton X-100, Tween 20, glycerol, DMEM, BSA (fraction V), and phloretin were from Sigma (St. Louis, MO). 2-deoxy-D-[3H]-glucose, A14-monoiodo-125I-labeled insulin, protein A-sepharose 6MB, nitrocellulose membrane (Hybond enhanced chemiluminescence), enhanced chemiluminescence kit containing secondary horseradish-labeled antibodies, and radiographs (Hyper film) were from Amersham Biosciences (Sao Paulo, SP, Brazil). Human recombinant regular insulin was from Biobrás (Montes Claros, Brazil). Pentobarbital sodium (Hypnol) was purchased from Cristalia, Quimicos (Itapira, Brazil). The glucose kit used for plasma glucose determination was from CELM (Glicose SL-E, Sao Paulo, Brazil), and the kit used for plasma insulin determination was from Linco Research (kit no. RI-13K, St. Charles, MO). Scintillation cocktail (Eco Lume) was from ICN Pharmaceuticals (Costa Mesa, CA). Bicinchoninic acid reagents were from Pierce Biotechnology (Rockford, IL).

Isolation of adipocytes.   The animals were killed (8:00 AM) under pentobarbital anesthesia (4 mg/100 g body wt) by decapitation after a 12-h fast, and the blood from the trunk was collected. The trained animals were killed 24 h after the last bout of exercise to prevent its acute effects. The sedentary rats were also killed at the same moment. Both epididymal fat pads were totally removed, weighed, minced with fine scissors, and digested at 37°C in a medium containing collagenase type II (1.25 mg/ml in 1 mM DMEM, 25 mmol HEPES, 4% BSA, pH 7.4), and the adipocytes were isolated according to Rodbell (31). The isolated adipocytes (~105 cell/ml) were suspended in Earle-HEPES-BSA (EHB) buffer, pH 7.4, at 37°C. Cell size and number were determined as previously described by Di Girolamo et al. (15).

Determination of the citrate synthase activity in soleus muscle.   Citrate synthase maximum activity was determined as previously described by Alp et al. (2). Briefly, under pentobarbital anesthesia (4 mg/100 g body wt), samples (100 mg) of soleus muscle were excised and homogenized twice (10 s each at maximum speed, Polytron PT 3100, Kinematica, Littau-Lucerne, Switzerland) in 1 ml of extraction buffer (50 mM Tris·HCl and 1 mM EDTA, pH 7.4) and centrifuged (3,800 g, 30 s, 4°C). The supernatant was used to analyze spectrophotometrically (412 nm) the citrate synthase (EC 4.1.3.7 [EC] ) maximum activity. The protein content was quantified using bicinchoninic acid reagents. The maximal enzyme activity was expressed as nanomoles per minute per milligram of protein.

Plasma glucose and insulin levels.   Plasma glucose was determined by enzymatic glucose-oxidase/peroxidase method (7) with a commercial kit. Insulin levels were determined by radioimmunoassay using a specific antibody against rat insulin. The intra-assay variability was <5%.

Insulin-stimulated 2-deoxy-D-[3H]-glucose uptake rates.   2-Deoxy-D-[3H]-glucose uptake (2DGU) experiments were performed as described elsewhere with some modifications (26, 27). Briefly, aliquots (100 µl) of isolated adipocytes (20% cell suspension) were transferred to plastic test tubes (17 x 100 mm) with or without insulin (10 nM) diluted in EHB buffer (pH 7.4, final reaction volume = 400 µl), and the cells were incubated for 30 min in a water bath at 37°C. At the end of the incubation period, a 40-µl aliquot of the reaction mixture was pipetted in a 2-ml plastic tube containing a 10-µl aliquot of 2-deoxy-D-[3H]-glucose (0.4 mM final concentration and 0.05 µCi/tube), and the uptake reaction was allowed for exactly 3 min. 2DGU was interrupted by adding 250 µl of ice-cold phloretin (0.3 mM in EHB). Next, 200 µl of this last mixture were transferred to microfuge tubes (400-µl capacity) layered with 200 µl of silicone oil (density = 0.963 mg/ml) and centrifuged (Microfuge E, Beckman Instruments, Palo Alto, CA) for 9 s at 15,000 rpm. The cell pellet on top of the oil layer was removed to vials containing 3 ml of scintillation cocktail, and the trapped radioactivity was measured in a liquid scintillation counter (Tri Carb 2100TR, Packard Instrument, Meriden, CT). Unspecific 2-deoxy-D-[3H]-glucose radiolabel trapping was determined in a parallel tube already prepared with 250 µl of ice-cold phloretin to stop transport reaction from the beginning. This value was discounted from the total trapping, and the resultant specific uptake was recalculated to be expressed as picomole per square centimeter of cell surface area, which is a good index of the glucose transporter population density present in adipocyte plasma membrane.

Insulin binding to cell surface receptors.   From the same 20% adipocyte suspension, 450-µl aliquots in EHB (pH 7.8) were transferred to 12 x 75 mm polypropylene test tubes prepared with a 10-µl mixture of A14-monoiodo-125I-labeled insulin (10,000 counts·min–1·tube–1) in the absence or presence of cold insulin (0.025, 0.1, 0.25, 1, 2.5, and 10 nM) in a 500-µl final reaction volume. This mixture was incubated for 180 min in a water bath at 16°C to prevent receptor internalization (29). The assay was interrupted by the centrifugation of 200-µl aliquots through silicone oil, and the radioactivity trapped in the cell pellets on the top of the oil layer was measured as described elsewhere (27). The plasma membrane receptor number was determined according to Scatchard (34).

Tissue extraction, immunoprecipitation, and immunoblotting.   These analyses were performed as described elsewhere (32, 36). Briefly, rats, under pentobarbital anesthesia (4 mg/100 g body wt), received an intravenous bolus injection of regular insulin (50 nmol·l–1·100 g body wt–1) through the abdominal vena cava. Immediately before the injection and 90 s after it (at this time point, the maximal insulin-induced phosphorylation response was obtained, as determined by a time-course study performed in our laboratory), the periepididymal fat pads were excised and processed for analysis of IR-{beta}, IRS-1, IRS-2, and Akt (in this case, we observed 2 peaks of insulin-induced Akt phosphorylation: at 90 s and at 5 min) studies. The fat was immediately homogenized in freshly prepared ice-cold buffer (1% Triton X-100, 100 mM sodium orthovanadate, 10 mM EDTA, 100 mM Tris, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride, and 0.01 mg/ml aprotinin, pH 7.5) using a Polytron homogenizer (PT 3100) set at maximum speed for 30 s. Insoluble material was removed by centrifugation (15,000 g) for 20 min at 4°C. Protein concentration was estimated by Biuret method (16). Aliquots of the resulting supernatants containing 3 mg of total protein were immunoprecipitated with anti-IR-{beta} subunit or anti-IRS-1 or anti-IRS-2 antibodies. The antibodies were added to homogenates and incubated overnight at 4°C, followed by the addition of protein A sepharose 6MB for 2 h. The mixture was centrifuged (15,000 g, for 15 min at 4°C), and the pellets were washed three times in ice-cold buffer (0.5% Triton X-100, 100 mM Tris, pH 7.4, 10 mM EDTA, and 2 mM sodium orthovanadate) and then resuspended in Laemmli sample buffer (0.1% bromophenol blue, 1 mM sodium phosphate, pH 7.0, 50% glycerol, and 10% SDS) and boiled for 5 min before SDS-PAGE (6.5% bis-acrylamide) in a miniature slab gel (Mini-Protean, Bio-Rad). Proteins in gel were electrophoretically transferred for 50 min (120 V) at 4°C to a nitrocellulose membrane. Nonspecific protein binding to the nitrocellulose was reduced by preincubating the membrane in blocking buffer [5% (wt/vol) nonfat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20] for 4 h at 4°C. The membranes were blotted with anti-phosphotyrosine (1 µg/ml, 1:400), anti-IR-{beta} (1:400), anti-IRS-1 (1:400), or anti-IRS-2 (1:400) antibodies diluted in blocking buffer (3% BSA instead of nonfat dry milk). To check whether PI3-K co-immunoprecipitated with IRS-1 and IRS-2, the membranes were washed (90 min) three times for stripping the first antibody using a buffer (pH 2.8) that contained 200 mM glycine, 200 mM NaCl, and 1 N NaOH and reblotted with an antibody against the anti-p85 regulatory subunit of PI3-K (1:5,000). For the determination of the phosphorylation of Akt, total extracts of periepididymal fat pads were prepared, and 75 µg of total protein were resolved by SDS-PAGE (8% bis-acrylamide), transferred to nitrocellulose membranes, and blotted with anti-phosphoserine (Ser473) Akt (1:1,000) antibody. Each membrane was subsequently incubated with a secondary horseradish-labeled antibody for 1 h followed by the addition of substrate reaction mix for a chemiluminescent detection of the protein specimen. Results were visualized using Amersham Hyper Film. Band intensities of the exposed X-ray films were quantified by optical densitometry (Scion Image Software 4.02, Scion, Frederick, MD). Results were normalized assuming the mean of densitometric measurements of bands from the fat tissue of sedentary animals before insulin stimulation as 1 arbitrary unit.

Data presentation and statistical analysis.   All data are expressed as means ± SE. Student's t-test for unpaired samples was used to test for differences between groups for all parameters. For insulin signaling studies, two-way ANOVA for repeated measures and Bonferroni post hoc tests were adopted. A P value of 0.05 was established as a fiducial limit of significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Body weight, body weight gain, epididymal fat pad weight, and adipocyte volume.   As shown in Table 1, final body weight, body weight gain (final body wt – initial body wt), and epididymal fat pad weight from both groups were not statistically different. However, adipocyte volume from trained rats decreased after 7 wk of training compared with sedentary rats.


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  		Table 1. General characterisitics of sedentary and trained rats

 
Citrate synthase activity in soleus muscle.   Citrate synthase activity in soleus muscle was taken as an index of training effectiveness (25). As Table 1 shows, citrate synthase activity increased 40% (P < 0.05) in trained rats compared with sedentary rats.

Plasma glucose and insulin levels.   Plasma glucose and insulin are presented in Table 1. Trained rats exhibited a 7% decrease (P < 0.05) in plasma glucose and a 42% reduction (P < 0.05) in insulin levels compared with sedentary rats.

Insulin-stimulated 2DGU rates.   The uptake of the nonmetabolizable glucose analog 2-deoxy-D-[3H]-glucose was measured in isolated fat cells from trained and sedentary rats, both in the absence or in the presence of 10 nM insulin. This modified hexose, like glucose, is transported through the cell membrane by the same saturable transport system and is phosphorylated by hexokinase but not further metabolized, accumulating as 2-deoxy-D-glucose-6-phosphate (30). As shown in Table 1, adipocytes from trained rats increased insulin-induced glucose uptake (90%; P < 0.05) compared with sedentary rats. Basal rates of glucose uptake were not affected by training.

Insulin binding to cell surface.   Table 1 shows the number of insulin receptors in fat cell surfaces estimated by Scatchard analysis (34). The receptor number did not show any statistical difference between the groups, indicating that the differences in 2DGU are probably influenced by postreceptor mechanisms.

IR-{beta}.   Periepididymal adipose tissues were excised before and after an intravenous insulin injection and subsequently homogenized in ice-cold extraction buffer (4°C). Western blot analysis of the immunoprecipitated IR-{beta} subunit was performed to quantify protein content and the degree of tyrosine phosphorylation (PY) of IR-{beta} (for further details, see MATERIAL AND METHODS) (Fig. 1). These parameters did not differ significantly between groups either before or after insulin stimulation.



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  		Fig. 1. Insulin receptor-{beta} subunit (IR-{beta}) protein content (A) and IR-{beta} tyrosine phosphorylation (PY; B) in epididymal fat pads from trained (T) and sedentary (S) age-matched rats. IB, immunoblot. Scanning densitometry was performed on autoradiographs from 6 experiments. Data are means ± SE. Results were normalized assuming the mean of densitometric measurements of bands from the fat tissue of sedentary animals before insulin stimulation as 1 arbitrary unit (AU). Bars are aligned with the respective blots at top.

 
IRS-1 and PI3-K association.   Figure 2A shows a representative blot of the immunoprecipitated IRS-1 protein content. There were no significant differences between the groups. Insulin-induced PY IRS-1 increased 57% (P < 0.05) in trained rats compared with sedentary rats (Fig. 2B). The following step in insulin signaling, the PY IRS-1 association to the regulatory p85 subunit of PI3-K, was also determined (Fig. 2C). After insulin injection, the adipose tissue from the trained rats showed a 1.57-fold increase (P < 0.05) in IRS-1/PI3-K association compared with sedentary rats.



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  		Fig. 2. Insulin receptor substrate (IRS)-1 protein content (A), IRS-1 tyrosine phosphorylation (B), and IRS-1/phosphatidylinositol 3 (PI3)-kinase (p85 subunit) association (C) in epididymal fat pads from T and S age-matched rats. Scanning densitometry was performed on autoradiographs from 6 experiments. Data are means ± SE. Results were normalized assuming the mean of densitometric measurements of bands from the fat tissue of S animals before insulin stimulation as 1 AU. Bars are aligned with the respective blots at top. *P < 0.05.

 
IRS-2 and PI3-K association.   Figure 3A shows a representative blot of the immunoprecipitated IRS-2 protein content. There were no significant differences between the groups. Our results show that trained rats increased basal (1.4-fold; P < 0.05) and insulin-stimulated PY IRS-2 (2.38-fold; P < 0.05) compared with sedentary rats (Fig. 3B). However, the insulin-induced IRS-2/PI3-K association was not significantly different in both groups (Fig. 3C).



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  		Fig. 3. IRS-2 protein content (A), IRS-2 tyrosine phosphorylation (B), and IRS-2/PI3-kinase (p85 subunit) association (C) in epididymal fat pads from T and S age-matched rats. Scanning densitometry was performed on autoradiographs from 6 experiments. Data are means ± SE. Results were normalized assuming the mean of densitometric measurements of bands from the fat tissue of S animals before insulin stimulation as 1 AU. Bars are aligned with the respective blots at top. *P < 0.05.

 
AKT.   To determine whether PI3-K downstream signaling was affected by training, we measured Akt serine473 phosphorylation (P-S473-Akt) in periepididymal fat pads excised before and 90 and 300 s after insulin injection (Fig. 4). After 90 s, the adipose tissue from trained rats showed a 3.15-fold increase (P < 0.05) in P-S473-AKT compared with the sedentary rats. However, after 5 min, there were no statistical differences between the groups.



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  		Fig. 4. Insulin-stimulated Akt serine473 phosphorylation in epididymal fat pad from T and S age-matched rats. Scanning densitometry was performed on autoradiographs from 6 experiments. Data are means ± SE. Results were normalized assuming the mean of densitometric measurements of bands from the fat tissue of S animals before insulin stimulation as 1 AU. Bars are aligned with the respective blots at top. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Endurance exercise training has been reported to increase insulin action in experimental models using animals or humans. This adaptation is particularly prevalent in muscle and white adipose tissue. At the adipocyte level, the improved insulin response is accompanied by a reduction in mean cell diameter after long-lasting exercise sessions in humans and animals (12, 13, 14). Endurance exercise-trained rats exhibited increased 2DGU compared with sedentary age-matched rats after insulin stimulation. Our findings are in accordance with previous reports indicating that chronic exercise induces an increase in insulin responsiveness not only in muscles but also in adipocytes that exhibited a reduction in size (11, 13, 14, 17, 39). Other researchers attributed this adaptation to an augment in insulin receptor numbers that was not observed in the present work. However, the training schedules adopted in those studies were considerably different since the animals were submitted to prolonged swimming exercise sessions (6 h/day) (14, 37). Furthermore, the augmentation (18%) in insulin binding was not enough to explain the intense increase (~4 times) in adipocyte insulin responsiveness (37). In conclusion, our result is in accordance with a previous study in which rats trained by treadmill running did not show any differences in insulin binding (38).

Intracellular events linking insulin binding to its receptor and the glucose uptake by GLUT4 translocation to plasma membrane have been studied in models of chronic and acute exercise and used to explain how insulin controls its biological effect (19, 21, 28). Here, we analyzed the early steps of insulin signaling in periepididymal fat pads from sedentary and trained rats in an attempt to elucidate the increased insulin responsiveness observed after exercise training. The first step of insulin signaling transduction pathway is the autophosphorylation of IR-{beta} after insulin binding to its extracellular {alpha}-subunit. Such a phenomenon triggers tyrosine kinase activation of IR-{beta} that phosphorylates a wide range of proteins in the cytosol and on the plasma membrane inner surface (33). In our study, we did not observe any differences in PY IR-{beta} and its protein content after 7 wk of physical training in rats. On the other hand, studies conducted in skeletal muscle homogenates have demonstrated controversial results about the effects of endurance exercise training on the first step of insulin signaling. For instance, Chibalin et al. (10) reported an increased IR-{beta} protein content and phosphorylation in skeletal muscle after 5 days of training in rats. However, Yu et al. (40) showed a reduction in protein content of IR-{beta} in vastus lateralis from endurance exercise runners. In our laboratory's previous work, we did not observe difference in PY IR-{beta} or protein content in rats after 6 wk of swimming training (28). Possibly, different schedules of training and species-specific or tissue-specific factors might justify some discrepancies found in these studies, indicating that further work is needed to clarify this issue.

As described above, regular exercise did not change the protein content of IRS-1 and IRS-2 after endurance exercise training, although several studies have demonstrated discrepancies, like reductions in IRS-1 and -2 (40), no differences (28), or augmented IRS-2 expression (10). We also studied the insulin-induced PY IRS-1 and PY IRS-2. Apart from protein content, chronic exercise was efficient to augment the phosphorylation of both IRS isoforms compared with sedentary animals after insulin stimulation. IRS-1 and -2 are key proteins in insulin-responsive cells because they link the membrane-generated signal to PI3-K, which activates this enzyme, which will ultimately recruit downstream proteins involved in the GLUT4 translocation from trans-Golgi to plasma membrane and subsequently increase glucose transport (22, 24). In our study, an increased insulin-stimulated IRS-1/PI3-K association was found in the trained group. Interestingly, the amount of association showed almost a stoichiometric correlation with glucose transport and PY IRS-1. Notwithstanding that basal and insulin-stimulated PY IRS-2 were increased in adipose tissue homogenates from trained rats, we observed only a slight tendency to increase the IRS-2/PI3-K association. IRS-2 may likely be associated with other docking proteins such as Grb2, which in conjunction with Shc, SOS, and Ras operates as a molecular switch stimulating the MAPK pathway and subsequent gene expression (33), but this was not explored here. In addition, it is important to note that only the association of IRS-1 and -2 to PI3-K does not reflect the activity of the enzyme, and consequently, IRS-2 must not be completely ruled out. Hence, the increased insulin responsiveness of adipocytes from endurance exercise-trained rats seems to be mediated preferentially by IRS-1 regardless of reports showing that adipocytes from IRS-2 knockout mice elicited a diminished glucose uptake and the overexpression of IRS-1 did not fully reestablish this insulin-mediated effect (6, 18). However, these two studies were not performed in exercised or trained mice. In another work with IRS-2 knockout mice subjected to an acute exercise, an increase in muscle glucose uptake was observed (20).

To examine more distal steps in insulin signaling through the PI3-K pathway, we determined the capacity of insulin to induce serine Akt phosphorylation (P-S473-Akt), a protein with a pleckstrin domain and pleiotropic actions. This downstream insulin effector is directly activated by a product of PI3-K, phosphotidylinositol-3-phosphate, and additionally modulated by its upstream activator phosphoinositide-dependent kinases (PDK)-1 and -2, and is an essential step in glucose uptake (1). We found increased P-S473-Akt after 90 s of insulin stimulation in trained rats compared with sedentary rats, but not after 5 min (300 s). As previously demonstrated, gastrocnemius from trained rats showed an increased P-S473-Akt after 5 min of insulin stimulation (28). In this work, Akt content in muscle did not change with training. Therefore, because our results show similar effects in white adipose tissue, we propose that Akt phosphorylation represents an important event to explain the increased insulin-induced glucose uptake of endurance exercised-trained rats.

The data observed in adipose tissue from trained rats are in agreement with our laboratory's previous report conducted in striated skeletal muscle showing that proteins involved in the first steps of insulin signal transduction mediate the increased insulin responsiveness (28). At least in muscles, the PI3-K/Akt pathway is an important contributor to the improvement in insulin-induced glucose transport after training. Although the same pathway seemed to be important in adipose tissue, alternative routes probably might be implicated. Recent data have proposed the involvement of atypical PKC-{lambda}/-{zeta} and the lipid raft complex as strong mediators (6, 35). It is important to note that this is the first experimental observation showing the effects of endurance exercise training through the first steps of insulin signaling in adipocytes from trained rats.

In summary, the results of our study show that 1) endurance exercise training improved insulin responsiveness in isolated adipocytes, 2) this adaptation is not a consequence of the increased insulin binding to adipocytes, and 3) this improvement in glucose uptake involves at least the IRS/PI3-K/Akt pathway, and it seems that IRS-1 (more than IRS-2) is preferentially implicated.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by Sao Paulo State Research Foundation Grant 99/09689-2.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. B. Lima, Prof. Lineu Prestes Ave., 1524-Dept. of Physiology and Biophysics, Institute of Biomedical Sciences (ICB 1 Bldg.), Univ. of Sao Paulo, 05508-900, Sao Paulo, Brazil (E-mail: fabio{at}icb.usp.br)

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
 

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