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Regulation of Cbl-Associated Protein/Cbl Pathway in Muscle and Adipose Tissues of Two Animal Models of Insulin Resistance

Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, 13081-970 Campinas, São Paulo, Brazil

Address all correspondence and requests for reprints to: Mario J. A. Saad, M.D., Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, 13081-970 Campinas, São Paulo, Brazil. E-mail: msaad{at}fcm.unicamp.br.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The phosphatidylinositol 3-kinase-independent pathway to induce glucose transport may involve the tyrosine phosphorylation of the protooncogene c-Cbl. In the present study, we examined whether acute exposure to insulin stimulates the tyrosine phosphorylation of Cbl and its association with Cbl-associated protein (CAP) in muscle and adipose tissue of rats in vivo. We report herein that insulin induces Cbl tyrosine phosphorylation and association with CAP in adipose tissue but not in muscle. We also examined the expression and tyrosyl-phosphorylation state of Cbl and CAP/Cbl association in adipose tissue of rats submitted to prolonged fasting and in monosodium glutamate (MSG)-insulin-resistant rats. An increase in Cbl phosphorylation is observed in the fat of MSG rats, parallel with an increase in association of CAP-Cbl as well as an augment in CAP and Cbl protein expression in the adipose tissue of these animals. These events are accompanied by a decrease in insulin-stimulated insulin receptor/ insulin receptor substrate (IRS)-1 tyrosine phosphorylation and an increase in the IRS-2/phosphatidylinositol 3-kinase/Akt/Foxo1 pathway. In adipocytes of fasted rats, there is a decrease in CAP and Cbl protein expression, insulin-induced Cbl phosphorylation, and the association with CAP. In parallel, there is also a decrease in the insulin receptor/IRSs/Akt/Foxo1 pathway. Thus, insulin is able to induce Cbl tyrosine phosphorylation and its association with CAP in the adipose tissue of normal rats. In addition, our data provide evidence that the CAP-Cbl pathway may have a role in the modulation of adiposity in fasting and in MSG-treated rats.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE INSULIN RECEPTOR is a tyrosine kinase that catalyzes the phosphorylation of several intracellular substrates, including the insulin receptor substrate (IRS) proteins, GAB-1, Shc, APS, p60^DOK, SIRPS, and c-Cbl (1, 2, 3, 4, 5, 6, 7). Each of these substrates recruits a distinct subset of signaling proteins containing Src homology 2 (SH2) domains, which interact specifically with sequences surrounding the phosphotyrosine residue (8). Phosphatidylinositol 3-kinase [PI(3)K], which is responsible for phosphorylating phosphatidylinositol (PI), PI-4-P, and PI-4,5-P₂, is one of the signaling molecules that is activated by binding IRS-1 and IRS-2 and has been implicated in linking the insulin receptor (IR) to glucose transport (9, 10). However, there is compelling evidence that activation of PI(3)K-dependent pathways is not sufficient to induce glucose transport. For example, dramatic overproduction of constitutively active mutants of PI(3)K can only partially stimulate the translocation of the insulin-responsive glucose transporter protein (11). Other growth factors and adhesion molecules that can activate PI(3)K and its downstream kinases have no effect on glucose transport or GLUT4 translocation (12, 13, 14). Furthermore, cell-permeable derivates of phosphatidylinositol 3,4,5-triphosphate can stimulate GLUT4 translocation only when cells are pretreated with insulin (15). These data suggest that insulin must generate at least two independent signals to stimulate glucose transport, one dependent on and another independent of PI(3)K.

The PI(3)K-independent pathway appears to involve tyrosine phosphorylation of the Cbl protooncogene (7). The phosphorylation of Cbl by insulin requires the Cbl-associated protein (CAP), an adapter molecule that binds to proline-rich sequences in Cbl through its carboxyl-terminal SH3 domain (16). On phosphorylation of Cbl, the CAP-Cbl complex dissociates from the IR and moves to a caveolin- enriched, Triton-insoluble membrane fraction (17). Translocation of phosphorylated Cbl recruits the adapter protein CrkII to the lipid raft via interaction of the SH2 domain of CrkII with phospho-Cbl (18). CrkII also forms a constitutive complex with the guanyl nucleotide-exchange protein C3G. Once translocated into lipid rafts, C3G comes into proximity with the G protein TC10 and catalyzes the exchange of GTP for GDP, resulting in the activation of the protein (18). The localization of TC10 in rafts is required for its activation by insulin (19). Once activated, TC10 seems to provide a second signal to the GLUT4 protein that functions in parallel with the activation of the PI(3)K pathway (18).

CAP is highly expressed, at the mRNA level, in tissues such as heart, kidney, liver, skeletal muscle, and adipose tissue (16). However, it is not known whether insulin induces the tyrosine phosphorylation of Cbl and whether this phosphorylated protein can associate with CAP in animal tissues. In this study, we examined whether acute exposure to insulin can stimulate the tyrosine phosphorylation of Cbl and its association with CAP in muscle and adipose tissue of rats in vivo. We also examined the tyrosyl-phosphorylation state of the Cbl and Cbl/CAP association after insulin stimulation in vivo and the levels of these proteins in adipose tissue of rats, submitted to prolonged fasting, that exhibit hypoinsulinemia and altered insulin action and in monoglutamate (MSG)-insulin-resistant rats, with hyperinsulinemia and increased adiposity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Male Wistar rats were provided by the State University of Campinas-Central Breeding Center (Campinas, Brazil). Anti-Cbl (Cbl), anti-CAP (CAP), antiphosphotyrosine (PY), anti-IR (IR) antibodies, anti-IRS-1, anti-IRS-2, anti-Akt (protein kinase B), anti-Foxo1, anti-GSK3, and anti-pGSK3 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-pAkt and anti-pFoxo1 antibodies were from Cell Signaling Technology (Beverly, MA). Human recombinant insulin (Humulin R) was purchased from Eli Lilly (Indianapolis, IN). MSG was from Sigma Chemical Co. (St. Louis, MO). Routine reagents were purchased from Sigma unless otherwise specified. ¹²⁵I-Protein A was from Amersham (Amersham, UK).

Animals
Six-week-old male Wistar rats were fed with standard rodent chow and water ad libitum. Food was withdrawn 12–14 h before the experiments. In the experiments of prolonged fasting, the rats were fed (controls) or fasted for 72 h. For the MSG-treated animals, male newborn Wistar rats received an sc injection of 4.0 g/kg body weight of MSG or hyperosmotic saline (1.25 g/kg body weight) (control) every other day for the first 10 d of life. Pups were weaned on the 21st day of life and had free access to standard rodent chow and water ad libitum up to the age of 10 wk. Food was withdrawn 12–14 h before the experiments. All experiments were approved by the Ethics Committee of the State University of Campinas.

Methods
Rats were anesthetized with sodium amobarbital (15 mg/kg body weight, ip), and were used 10–15 min later, i.e. as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the cava vein exposed, and 0.5 ml normal saline or insulin were injected at a dose that was varied from 0.002 to 2 µg. At 1, 2, 3, 5, and 10 min after insulin injection, muscle and adipose tissues were removed, minced coarsely, and homogenized immediately in extraction buffer [1% Triton X-100, 100 mM Tris (pH 7.4) containing 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride, and 0.1 mg aprotinin/ml] at 4 C with a Polytron PTA 20S generator (model PT 10/35, Brinkmann Instruments, Westbury, NY) operated at maximum speed for 30 sec. The extracts were centrifuged at 15,000 rpm and 4 C in a 70.1 Ti rotor (Beckman, Palo Alto, CA) for 45 min to remove insoluble material, and the supernatants of these tissues were used for immunoprecipitation with IR, IRS-1, IRS-2, Cbl, and Protein A Sepharose 6 MB or Protein A/G plus (Santa Cruz Biotechnology).

Protein analysis by immunoblotting
The precipitated proteins and whole-tissue extracts were treated with Laemmli sample buffer (20) containing 100 mM dithiothreitol and heated in a boiling water bath for 4 min after which they were subjected to SDS-PAGE (6% bisacrylamide) in a miniature slab gel apparatus (Mini-Protean, Bio-Rad Laboratories, Hercules, CA). For total extracts, similar-sized aliquots (200 µg protein) were subjected to SDS-PAGE.

Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant) in a Bio-Rad miniature transfer apparatus (Mini-Protean) as described by Towbin et al. (21) except for the addition of 0.02% sodium dodecyl sulfate to the transfer buffer to enhance the elution of high-molecular-mass proteins. Nonspecific protein binding to the nitrocellulose was reduced by preincubating the filter overnight at 4 C in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose blot was incubated with the indicated antibodies, diluted in blocking buffer (0.3% BSA instead of nonfat dry milk) overnight at 4 C and then washed for 60 min with blocking buffer without milk. The blots were subsequently incubated with 2 µCi of ¹²⁵I-protein A (30 µCi/µg) in 10 ml blocking buffer for 2 h at room temperature and then washed again for 30 min as described above. ¹²⁵I-Protein A bound to the antiphosphotyrosine and antipeptide antibodies was detected by autoradiography using preflashed XAR film (Kodak, Rochester, NY) with Cronex Lightning Plus intensifying screens at -80 C for 12–48 h. Band intensities were quantitated by optical densitometry (Hoefer Scientific Instruments, San Francisco, CA; model GS 300) of the developed autoradiographs.

Insulin tolerance test
MSG and control (fasted overnight) fed and 72-h-fasted rats were submitted to an iv insulin tolerance test (ITT; 1 U/kg body weight of insulin), and samples for blood glucose measurements were collected at 0 (basal), 4, 8, 12, and 16 min after injection. Rats were anesthetized with sodium amobarbital as described above, 40 µl of blood were collected from their tails, and blood glucose concentration was measured by the glucose oxidase method. Thereafter, the rate constant for plasma glucose disappearance (K_itt) was calculated using the formula 0.693/(t_1/2). The plasma glucose t_1/2 was calculated from the slope of the least squares analysis of the plasma glucose concentrations during the linear phase of decline (22).

Histological analysis
Epididymal fat was completely removed and fixed in 10% paraformaldehyde solution. Thereafter, specimens were embedded in paraffin and mounted in glass slides in consecutive 10-µm sections. Staining was performed with hematoxylin and eosin. The mean adipose cell size was obtained using the Optomax image analysis system (Optomax, Burlington, MA).

Other
Plasma glucose was measured with a glucometer (Glucometer 4, Ames, Elkhart, IN), and serum insulin was detected by RIA, using a guinea pig antirat insulin antibody and rat insulin as standard (23). Serum-free fatty acid levels were analyzed in fed and 72-h-fasted rats and 12-h-fasted control and MSG rats using the NEFA-kit-U (Wako Chemical GmBH, Neuss, Germany) with oleic acid as a standard. Body lipid content was determined by a modification of the method of Leshner et al. (24). After shaving and removal of the gastrointestinal tract, carcasses were warmed overnight at 45 C. Softened carcasses were then homogenized in a blender. Lipid was extracted from 5-g aliquots with petroleum ether and determined gravimetrically (25). For measurement of muscle glycogen, gastrocnemius muscles were dissolved in 30% KOH and 5% Na₂SO₄ at 70 C for 15 min. Glycogen was then precipitated by mixing with 3 x volume of absolute alcohol and stored at -20 C overnight. The precipitates were collected by centrifugation at 13,000 x g for 5 min. The glycogen was hydrolyzed in 6 N H₂SO₄ at 100 C for 45 min and cooled. Samples were neutralized with 1 N NaOH, and glucose was measured using the glucose (HK) reagent (Sigma).

Statistical analysis
All groups of animals were studied in parallel. Comparisons between different groups were performed by employing t test for unpaired samples and ANOVA as appropriate. The level of significance adopted was P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cbl tyrosyl phosphorylation and its association with CAP and IR/CAP association after insulin stimulation in adipose tissue and muscle of rats
To determine tissue distribution of CAP and Cbl, we assayed whole-tissue extracts from fat and muscle. Using lysates with the same amount of protein, we performed immunoblotting with anti-Cbl (Fig. 1A) and anti-CAP (Fig. 1B) antibodies. Cbl protein expression was higher in adipose tissue than in muscle. Analyzing CAP expression, we observed a high expression of this protein in adipose tissue. However, we could not detect the presence of CAP in the muscle of rats.

View larger version (53K): [in this window] [in a new window]   FIG. 1. Time course and dose response of insulin-induced Cbl tyrosine phosphorylation and association with CAP. Whole-tissue extracts of fat and muscle were prepared as described in Materials and Methods and also using whole- tissue extract that was not centrifuged and dissolved directly in Laemmli sample buffer (A and B, right panel). Immunoblotting was performed with Cbl (A) or CAP (B) antibodies. Fat extracts from rats injected with saline (0) or insulin (1, 2, 3, 5, 10 min) or 5 min after different doses of insulin were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with IR antibody and immunoblotted with PY (C) or CAP antibody (D). Tissue extracts were also immunoprecipitated with Cbl antibody and immunoblotted with PY (E) or CAP (F) antibody. The results are representative of four different experiments.

To evaluate insulin-induced IR tyrosine phosphorylation, we performed experiments using samples from adipose tissue, immunoprecipitated with IR antibody, and immunoblotted with PY antibody. As shown in Fig. 1C, there is an increase in IR tyrosyl phosphorylation within 1 min after insulin stimulation, which was maximal at 2 min and almost vanished at 10 min after insulin administration. The insulin-stimulated IR phosphorylation in adipose tissue extracts was dose dependent (Fig. 1C). The presence of IR phosphorylation was detectable after the injection of 20 µg insulin, whereas maximal stimulation was observed with 2 µg insulin.

To examine whether CAP could form a complex with the IR, rats were stimulated with insulin for the indicated times and lysates from adipose tissue were immunoprecipitated with IR antibodies. The resulting immunoprecipitates were separated by SDS-PAGE and were analyzed by immunoblotting with CAP antibodies. As shown in Fig. 1D, the addition of insulin caused a time-dependent association of the CAP/IR complex. After 1 min of insulin stimulation, CAP was detected in the IR immunoprecipitate. The association was maximal 5 min after insulin infusion and almost vanished within 10 min. The insulin-stimulated IR/CAP association in adipose tissue extracts was dose dependent (Fig. 1D, bottom panel). The presence of IR/CAP association was detectable after the injection of 20 µg insulin and half- maximal stimulation occurred with 200 µg of the hormone. Maximal stimulation was observed with 2 µg insulin.

To investigate whether Cbl would undergo tyrosyl phosphorylation after stimulation by insulin, we infused insulin into the cava vein of 12 h fasted rats and then removed and homogenized the adipose tissue and immunoprecipitated the proteins with Cbl. These immunoprecipitates were analyzed for tyrosyl phosphorylation by immunoblotting with PY (Fig. 1E, upper panel). The presence of phosphorylated Cbl was detectable 1 min after insulin infusion and was maximal (12-fold above basal) 2–3 min after insulin injection, with a reduction thereafter. The insulin-stimulated phosphorylation of Cbl, as determined by Cbl immunoprecipitates of adipose tissue extracts, was dose dependent (Fig. 1E, bottom panel), with similar behavior of IR phosphorylation. Although we could not determine cava insulin levels, in preliminary experiments peripheral insulin levels obtained 90 sec after an intracava injection of 200 µg of insulin ranged between 40 and 70 µU/ml, which is similar to the normal physiological postprandial range in rats.

To gain further insight in the CAP-Cbl association, we analyzed the effect of insulin stimulation on the in vivo interaction between CAP and Cbl in adipose tissue of rats. When blots, previously analyzed with PY antibody, were subsequently reblotted with antibodies against CAP, we observed that the intensity of the bands increased after insulin stimulation. (Fig. 1F, upper panel). The presence of phosphorylated CAP/Cbl association was detectable 1 min after insulin infusion and was maximal 3 min after insulin infusion (Fig. 1F, upper panel). The dose-response relationship for the CAP/Cbl association in adipose tissue was similar to that of Cbl phosphorylation. Figure 1F, bottom panel, shows such a relationship and indicates that maximal effect was observed at 2 µg insulin.

To identify whether the same effects of insulin, through Cbl signaling pathways, could be observed in muscle, solubilized proteins from muscle stimulated with insulin were immunoprecipitated with Cbl antibody and subsequently incubated for different times with PY. We could not detect any tyrosine phosphorylation of Cbl protein in insulin-stimulated lysates from muscle of rats (data not shown).

The metabolic characteristics of MSG-treated rats
MSG rats had higher basal hyperinsulinemia (C: 40.0 ± 4.0 µU/ml vs. MSG: 67.0 ± 5.3 µU/ml; P < 0.05) but normal plasma glucose levels (C: 96 ± 2 mg/dl vs. MSG: 102 ± 1 mg/dl). MSG animals were more insulin resistant than the control rats, as expressed by their lower plasma glucose disappearance rates measured by the ITT (K_itt C: 4.51 ± 0.57%/min vs. MSG: 2.37 ± 0.41%/min; P < 0.05). Muscle glycogen stores were lower in MSG rats, compared with controls (C: 0.46 ± 0.05 mg/100 g tissue vs. MSG: 0.24 ± 0.04 mg/100 g tissue; P < 0.01). Free fatty acids were slightly, but not statistically significantly, higher in MSG rats than in control animals (C: 0.42 ± 0.05 µmol/ml vs. MSG: 0.46 ± 0.04 µmol/ml). The mean body weight of MSG rats was decreased when compared with controls (C: 306 ± 5 g vs. MSG: 238 ± 5 g; P < 0.05). Despite this finding, the relative weight of the epididymal fat pad of MSG rats was significantly higher (C: 1.00 ± 0.02 g/100 g body weight vs. MSG: 2.00 ± 0.04 g/100 g body weight; P < 0. 05), and the body lipid content was also higher when compared with controls (C: 10.5 ± 0.5 vs. MSG: 29.0 ± 1.0; P < 0.05). Histological analysis revealed that mean epididymal fat cell volume doubled in MSG rats (C: 1338 ± 45 µm² vs. MSG: 2.830 ± 191 µm²; P < 0.01), whereas cell density was reduced by about 40% in MSG rats.

Tyrosine phosphorylation of IR and downstream signaling in muscle of MSG rats
Insulin-induced IR tyrosine phosphorylation was reduced in the muscle of MSG animals (C: 100 ± 6 vs. MSG: 67 ± 9; P < 0.05, Fig. 2A). There was no difference in IR protein expression in the muscle of control and MSG rats (Fig. 2B).

View larger version (37K): [in this window] [in a new window]   FIG. 2. Insulin signaling in muscle of controls and MSG-treated rats. Muscle extracts from rats injected with saline (-) or insulin (+) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with IR antibody and immunoblotted with PY antibody (A). Tissue extracts were also immunoprecipitated with anti-IRS-1 and anti-IRS-2 antibodies and immunoblotted with PY (C and E) or anti-PI(3)K antibodies (D and F). Whole-tissue extracts were immunoblotted with IR (B), anti-phospho Akt (G), anti-Akt antibodies (H), anti-phospho-GSK3 (I), or anti-GSK3 (J). The results of scanning densitometry (n = 6) were expressed as arbitrary units. Columns and bars represent the mean ± SEM. *, P < 0.05, insulin control vs. insulin MSG.

Insulin-induced IRS-2 tyrosine phosphorylation was decreased in muscle, compared with controls (C: 100 ± 5 vs. MSG: 58 ± 6; P < 0.05, Fig 2E), as was IRS-2/PI(3)K association (C: 100 ± 5 vs. MSG: 75 ± 6; P < 0.05, Fig. 2F). There were no differences in IRS-2 protein levels in MSG rats, compared with controls (data not shown).

In MSG animals, insulin-induced Akt phosphorylation was significantly lower than controls (C: 100 ± 7 vs. MSG: 78 ± 4, P < 0.05, Fig. 2G). There were no differences in Akt protein levels in MSG rats, compared with controls (Fig. 2H).

Furthermore, in MSG animals, insulin-induced GSK3 phosphorylation was significantly lower than that of controls (C: 100 ± 8 vs. MSG: 65 ± 6, P < 0.05, Fig. 2G). There were no differences in GSK3 protein expression in MSG rats, compared with controls (Fig. 2H).

Cbl tyrosyl phosphorylation and CAP/Cbl association in adipose tissue of MSG rats
Insulin-induced IR tyrosine phosphorylation was reduced in adipose tissue of MSG animals (C = 100 ± 10 vs. MSG = 72 ± 8; P < 0.05; Fig. 3A). There was no difference in the IR protein expression in the adipose tissue of control rats and MSG rats (C = 100 ± 9 vs. MSG = 97 ± 12; P = 0.8; Fig. 3B).

View larger version (35K): [in this window] [in a new window]   FIG. 3. Insulin-induced Cbl tyrosine phosphorylation and association with CAP in adipose tissue of controls and MSG-treated rats. Fat extracts from rats injected with saline (-) or insulin (+) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with IR antibody and immunoblotted with PY antibody (A). Tissue extracts were also immunoprecipitated with Cbl antibody and immunoblotted with PY (C) or CAP (D). Whole-tissue extracts were immunoblotted with anti-IR (B), Cbl (E), and CAP (F) antibodies. The results of scanning densitometry (n = 6) were expressed as arbitrary units. Columns and bars represent the mean ± SEM. *, P < 0.05, insulin control vs. insulin MSG. #, P < 0.05, vehicle lean vs. vehicle obese. *, P < 0.05, insulin lean vs. insulin obese.

We then investigated the levels of Cbl protein by immunoblotting membranes containing transferred adipose tissue extracts with the antibody against Cbl. Using this approach, the adipose tissue level of Cbl was found to be increased in MSG rats, compared with control rats (C = 100 ± 3 vs. MSG = 123 ± 1; P < 0.05; Fig. 3E). To better define the level of CAP, an immunoblot with a specific antibody against this substrate was performed. Tissue expression of CAP was increased in adipose tissue of MSG rats, compared with control rats (C = 100 ± 9 vs. MSG = 152 ± 9; P < 0.05; Fig. 3F).

Insulin signaling in adipocytes of MSG rats
There was a significant decrease in insulin-stimulated IRS-1 tyrosine phosphorylation in MSG when compared with controls (C: 100 ± 9 vs. MSG: 70 ± 10; P < 0.05; Fig. 4A), without changes in IRS-1 protein expression (data not shown). When we analyzed IRS-1/PI(3)K association, in MSG rats there was a significant decrease when compared with controls (C: 100 ± 8 vs. MSG: 64 ± 7; P < 0.05; Fig. 4B).

View larger version (35K): [in this window] [in a new window]   FIG. 4. Insulin signaling in adipose tissue of controls and MSG-treated rats. Fat extracts from rats injected with saline (-) or insulin (+) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with anti-IRS-1 antibody and immunoblotted with PY (A) or anti-PI(3)K antibodies (B). Tissue extracts were also immunoprecipitated with anti-IRS-2 antibody and immunoblotted with PY(C) or anti-PI(3)K antibodies (D). Whole-tissue extracts were immunoblotted with anti-PI(3)K (E), anti-phospho Akt (F), anti-Akt (G) antibodies, anti-phosphoFoxo1 (H), and anti-Foxo1 (I). The results of scanning densitometry (n = 6) were expressed as arbitrary units. Columns and bars represent the mean ± SEM. #, P < 0.05, vehicle control vs. vehicle MSG. *, P < 0.05, insulin control vs. insulin MSG.

In MSG animals, insulin-induced Akt phosphorylation was significantly higher, compared with controls (C: 100 ± 8 vs. MSG: 228 ± 22, P < 0.05; Fig. 4F), without changes in Akt protein expression (Fig. 4G).

Basal Foxo1 phosphorylation was higher in MSG animals (C: 35 ± 4 vs. MSG: 61 ± 6, P < 0.05; Fig. 4H). Insulin-induced Foxo1 phosphorylation was also significantly higher in MSG than in control rats (C: 100 ± 11 vs. MSG: 206 ± 29, P < 0.05; Fig. 4H), without changes in Foxo1 protein expression (Fig. 4I).

The metabolic characteristics of 72-h-fasted rats
Rats starved for 72 h showed a decrease in body weight from 211 ± 62 to 165 ± 8 g (P < 0.05) and a parallel decrease in the levels of plasma glucose (fed = 165 ± 8 vs. fasted = 74 ± 2 mg/dl; P < 0.05) and serum insulin (fed = 38 ± 4. vs. fasted = 13 ± 4 µU/ml; P < 0.05). Seventy-two-hour-fasted animals were more insulin resistant than fed rats, as expressed by their lower plasma glucose disappearance rates measured by the ITT (K_itt fed: 4.22 ± 0.52%/min vs. fasted: 2.88 ± 0.36%/min; P < 0.05). Muscle glycogen stores were lower in fasted rats, compared with controls (fed: 0.52 ± 0.08 mg/100 g tissue vs. fasted: 0.31 ± 0.06 mg/100 g tissue; P < 0.01). Free fatty acids were higher in fasted rats than in control animals (fed: 0.35 ± 0.04 µmol/ml vs. fasted: 0.74 ± 0.07 µmol/ml). The relative weight of epididymal fat pad of 72-h-fasted-rats was significantly lower (fed: 0.76 ± 0.05 g/100 g body weight vs. fasted:0.49 ± 0.04 g/100 g body weight; P < 0.01). Histological analysis revealed that after 3 d of fasting, there was a reduction of about 50% in fat cell size (fed: 1026 ± 58 µm² vs. fasted: 580 ± 83 µm²; P < 0.01) and an increase of about 90% in cell density.

Insulin signaling in the muscle of fasted rats
Insulin-induced IR tyrosine phosphorylation was increased in the muscle of fasted animals (fed: 100 ± 6 vs. fasted: 208 ± 19; P < 0.01, Fig. 5A). There was no difference in the IR protein expression in muscle of fed rats and fasted rats (Fig. 5B).

View larger version (37K): [in this window] [in a new window]   FIG. 5. Insulin signaling in muscle of fed and fasted rats. Muscle extracts from rats injected with saline (-) or insulin (+) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with IR antibody and immunoblotted with PY antibody (A). Tissue extracts were also immunoprecipitated with anti-IRS-1 and anti-IRS-2 antibodies and immunoblotted with PY (C and E) or anti-PI(3)K antibodies (D and F). Whole-tissue extracts were immunoblotted with anti-IR (B), anti-phospho Akt (G), anti-Akt antibodies (H), anti-phospho-GSK3 (I), or anti-GSK3 (J). The results of scanning densitometry (n = 6) were expressed as arbitrary units. Columns and bars represent the mean ± SEM. *, P < 0.05, insulin fed vs. insulin fasted.

Insulin-induced IRS-2 tyrosine phosphorylation was increased in the muscle of fasted rats, compared with fed rats (fed: 100 ± 9 vs. fasted: 176 ± 19; P < 0.01; Fig. 5E), and IRS-2/PI(3)K association was also increased (fed: 100 ± 11 vs. fasted: 181 ± 26; P < 0.05; Fig. 5F). There were no differences in IRS-2 protein levels in fasted rats, compared with fed controls (data not shown).

In fasted animals, insulin-induced Akt phosphorylation was higher, compared with that of controls (fed: 100 ± 6 vs. fasted: 147 ± 9, P < 0.05; Fig. 5G). There were no differences in Akt protein levels in fasted rats, compared with fed controls (Fig. 5H).

In addition, in fasted animals, insulin-induced GSK3 phosphorylation was higher, compared with controls (fed: 100 ± 4 vs. fasted: 154 ± 11, P < 0.05; Fig. 5I). There were no differences in Akt protein levels in fasted rats, compared with fed controls (Fig. 5J).

Effect of fasting on Cbl tyrosyl phosphorylation and Cbl/CAP association in adipose tissue of fasted rats
In adipose tissue, insulin-induced IR phosphorylation was reduced in fasted rats when compared with the fed controls (fed = 100 ± 9 vs. fasted = 64 ± 8; P < 0.05; Fig. 6A). There were no differences in the IR levels in the adipose tissue of fasted rats when compared with their controls (fed = 100 ± 10 vs. fasted = 88 ± 12; P = 0.452; Fig. 6B).

View larger version (36K): [in this window] [in a new window]   FIG. 6. Insulin-induced Cbl tyrosine phosphorylation and association with CAP in adipose tissue of fed and fasted rats. Fat extracts from rats injected with saline (-) or insulin (+) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with IR antibody and immunoblotted with PY antibody (A). Tissue extracts were also immunoprecipitated with Cbl antibody and immunoblotted with PY (C) or CAP (D). Whole-tissue extracts were immunoblotted with anti-IR (B), Cbl (E), and CAP (F) antibodies. The results of scanning densitometry (n = 6) were expressed as arbitrary units. Columns and bars represent the mean ± SEM. *, P < 0.05, insulin fed vs. insulin fasted.

Cbl protein expression was decreased in the adipose tissue of fasting rats, compared with fed animals (fed =100 ± 18 vs. fasted = 40 ± 8; P < 0.05; Fig. 6E). The expression CAP was also found to be decreased in the adipose tissue of fasting rats, compared with fed animals (fed = 100 ± 7 vs. fasted = 52 ± 5; P < 0.01; Fig. 6F).

Insulin signaling in adipocytes of fasted rats
There was a significant decrease in insulin-stimulated IRS-1 tyrosine phosphorylation in fasted rats when compared with their controls (fed: 100 ± 9 vs. fasted: 54 ± 12; P < 0.05; Fig. 7A). When we analyzed IRS-1/PI(3)K association, in fasted rats there was a significant decrease when compared with controls (fed: 100 ± 8 vs. fasted: 49 ± 7; P < 0.05; Fig. 7B). There was also a decrease in IRS-1 protein expression in the adipose tissue of fasted rats (fed: 100 ± 6 vs. fasted: 71 ± 12; P < 0.05; data not shown)

View larger version (33K): [in this window] [in a new window]   FIG. 7. Insulin signaling in adipose tissue of fed and fasted rats. Fat extracts from rats injected with saline (-) or insulin (+) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with anti-IRS-1 antibody and immunoblotted with PY (A) or anti-PI(3)K antibodies (B). Tissue extracts were also immunoprecipitated with anti-IRS-2 antibody and immunoblotted with PY (C) or anti-PI(3)K antibodies (D). Whole-tissue extracts were immunoblotted with anti PI(3)K (E), anti-phospho Akt (F), anti-Akt (G) antibodies, anti-phospho-Foxo1 (H), and anti-Foxo1 (I). The results of scanning densitometry (n = 6) were expressed as arbitrary units. Columns and bars represent the mean ± SEM. #, P < 0.05, vehicle control vs. vehicle MSG. *, P < 0.05, insulin fed vs. insulin fasted.

In the adipose tissue of fasted animals, insulin-induced Akt phosphorylation was significantly lower when compared with their controls (fed: 100 ± 7 vs. fasted: 58 ± 11, P < 0.05; Fig. 7F), without changes in Akt protein expression (Fig. 7G).

Basal Foxo1 phosphorylation was higher in fed animals (fed: 32 ± 8 vs. MSG: 11 ± 3, P < 0.05; Fig. 7H). However, insulin-induced Foxo1 phosphorylation was significantly lower in fasted rats than in fed rats (fed: 100 ± 11 vs. fasted: 53 ± 9, P < 0.05; Fig. 7H), without changes in Foxo1 protein expression (Fig. 7I).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies have demonstrated a second pathway of insulin signaling required for GLUT4 translocation and glucose uptake that could proceed through a signaling pathway originating from lipid rafts and resulting in the ultimate activation of a small G protein, TC10 (26). A central figure in this pathway is CAP (16). In most insulin-responsive cells, Cbl is associated with the adapter protein CAP (27). We report here that Cbl is markedly and rapidly tyrosine phosphorylated in adipose tissue in response to insulin. Moreover, tyrosine-phosphorylated Cbl associates with CAP, and there is an increase in this association after insulin stimulation. In contrast, in rat muscle we could not detect tyrosine phosphorylation of Cbl, which may be due to the very low or undetectable amount of CAP protein in muscle, even though CAP mRNA has earlier been described by others to be expressed in this tissue (16). Thus, our study demonstrates that, in rats, the insulin-induced tyrosine phosphorylation of Cbl takes place in adipose tissue but not in muscle.

To gain further insight into the physiological role of the insulin induced CAP-Cbl complex in fat, we investigated the regulation of the CAP-Cbl pathway and the classic pathway of insulin signaling [IRSs/PI(3)K/Akt pathway] in the adipose tissue of two animal models of insulin resistance with different total mass of adipose tissue: MSG rats with increased adiposity and insulin resistance and 72-h-fasted rats with decreased adiposity and also with insulin resistance.

Early postnatal administration of MSG in rats induces increased adiposity, hyperinsulinemia and insulin resistance. Our results show that, in MSG insulin-resistant rats, there is an important decrease in insulin-stimulated IRS/PI(3)K/Akt pathway in muscle, which may have an important role in the insulin resistance of those animals. Insulin stimulates glycogen accumulation through a coordinated increase in glucose transport and glycogen synthesis. The hormone activates glycogen synthase by promoting its dephosphorylation through the inhibition of kinases such as protein kinase A or GSK-3 (28) and activation of protein phosphatase 1 (29). Upon its activation downstream of PI(3)K, Akt phosphorylates and inactivates GSK-3, decreasing the rate of phosphorylation of glycogen synthase, thus increasing its activity state (28). Our results showing an increase in GSK-3 in muscle of MSG rats are in accordance with a decreased Akt phosphorylation and also in glycogen content in these animals.

Analogously, in fat we observed a decrease in insulin-stimulated IR tyrosine phosphorylation that is accompanied by a decrease in the IRS-1 pathway and, surprisingly, by an increase in IRS-2 tyrosine phosphorylation and PI(3)K association that lead to an increase in Akt serine phosphorylation and in Foxo1 phosphorylation. Furthermore, we observed an increase in Cbl phosphorylation, which parallels the increase in the association of CAP-Cbl as well as an augmentation in CAP and Cbl protein expression in the adipose tissue of these animals.

Thus, insulin resistance in the IR/IRS-1/PI(3)K pathway contrasts markedly with the ability of insulin to stimulate the IRS-2/PI(3)K and CAP/Cbl pathways in the adipose tissue of obese MSG rats. Insulin resistance does not affect the IRS-2/PI(3)K and CAP-Cbl pathways because insulin administration increased IRS-2 and Cbl phosphorylation to a greater extent in MSG-treated rats than controls. The differential regulation of IRS-1 and IRS-2 was also reported in adipocytes of noninsulin-dependent diabetes mellitus. In normal human adipocytes, IRS-1 has been demonstrated as the main docking protein for PI(3)K with an associated increase in glucose uptake. However, in type 2 diabetes IRS-2 is able to replace IRS-1 as the main docking protein for binding and activation of PI(3)K (30). The reasons for this specific modulation are unknown, but some possibilities should be considered. We previously demonstrated that, in MSG animals, there is an increase in protein tyrosine phosphatase 1b expression and association with IR in adipose tissue (31). Because protein tyrosine phosphatase 1b can also interact with and dephosphorylate IRS-1 (32), we suggest that this is a possible mechanism to decrease IR and IRS-1 tyrosine phosphorylation while preserving the IRS-2/PI3k/Akt and CAP/Cbl pathways. Another possibility that should not be excluded is an increase in the serine phosphorylation level of IR and IRS-1, without a similar effect on IRS-2.

The alteration in the early steps in insulin signaling in the muscle of MSG rats is probably not related to free fatty acid levels because this metabolic substrate is similar in MSG and control rats. The absence of free fatty acid elevation in MSG rats despite the increase in adipose mass may be a consequence of an increased IRS-2/PI3K pathway in this tissue, which may mediates insulin’s antilipolytic effect.

According to previous reports, MSG-insulin-resistant rats demonstrate hypersensitivity to insulin in isolated adipocytes, reflected by increased insulin-stimulated glucose transport and lipogenic activity (33). Taking together with the present data and studies on glucose transport in adipocytes of MSG rats (33), it may be suggested that the increase in Cbl tyrosyl phosphorylation and CAP/Cbl association in addition to the IRS-2/PI(3)K/Akt pathway play an important role in the increased glucose transport described. Furthermore, Akt phosphorylates the Foxo transcription factors (34) and inhibits their transcriptional activity (35, 36). Foxo1 is the most abundant Foxo isoform in several insulin-responsive tissues, such as liver, adipose tissue, and the pancreatic ß-cell. Recently Foxo1 has been shown to play an important role in coupling insulin signaling to adipocyte differentiation (37). The increased activation of Akt in the adipose tissue of MSG rats is associated with increased phosphorylation and inactivation of Foxo1, leading to enhanced adipogenesis.

Prolonged fasting in rats is characterized by insulin deficiency and insulin resistance (38, 39). Both peripheral and hepatic insulin resistance to glucose metabolism have been observed in vivo (40). However, the insulin resistance of fasted animals, characterized by a reduced glucose disappearance rate during ITT, associated with reduced glycogen content in muscle and high levels of free fatty acids, may be downstream of IRSs/PI(3)K/AKT/GSK in muscle.

In adipocytes, there was a decrease in the IR/IRSs/Akt pathway, and in parallel there was also a decrease in CAP and Cbl protein expression and in insulin-induced Cbl phosphorylation and association with CAP. These alterations in the early steps of insulin action in adipocytes of 72-h-fasted rats may contribute to explain previous reports of reduced glucose uptake in the adipose tissue of these animals (41, 42). Additionally, in fasted rats, reduced Akt activation associated with reduced inactivation of Foxo1 might explain the lack of increased adiposity.

The modulation of CAP/Cbl in 72-h-fasted-rats contrasts with the regulation of these proteins in adipose tissue of MSG-treated rats. It is interesting to note that in the MSG animal, with an increase in adiposity and glucose uptake in this tissue, there was an up-regulation of CAP and Cbl. In 72-h-fasted rats, which have decreased adipose tissue and decreased glucose uptake in their adipocytes, we observed a reduction in these proteins.

An interesting finding, in these two animal models, was the parallelism between insulin levels and the regulation of Cbl protein expression, tyrosine phosphorylation, and association with CAP. In the animal model with hyperinsulinemia, there was an increase in the CAP/Cbl pathway, and in the hypoinsulinemic model, there was a decrease in this pathway. This regulation is not accompanied by IR or IRS-1 tyrosine phosphorylation in MSG rats. This implies that, in adipose tissue, a clear dissociation between IR tyrosine phosphorylation and Cbl-tyrosine phosphorylation and association with CAP is possible and may contribute to explain the increase in adiposity in this animal despite the reduced molecular activation of the IR/IRS-1 pathway.

In conclusion, our results demonstrate that insulin is able to induce Cbl-tyrosine phosphorylation and association with CAP in adipose tissue of intact rat in a dose-dependent manner. MSG-treated rats and fasting can modulate these proteins, and this modulation parallels the plasma insulin level, which may play a role in the increased adiposity. In addition, our data provide direct evidence that the CAP-Cbl pathway is not required for insulin signaling in the muscle of rats and that, in an insulin-resistant state, the IRS-2/PI(3)K and CAP/Cbl pathway appears to be selectively enhanced in the adipose tissue of MSG-treated rats.

	Footnotes

 
A.C.P.T. and J.B.C.C. contributed equally to this paper.

Abbreviations: Akt, Protein kinase B; Cbl, anti-Cbl; CAP, c-Cbl-associating protein; CAP, anti-CAP; IR, insulin receptor; IR, anti-IR; IRS, insulin receptor substrate; ITT, insulin tolerance test; K_itt, rate constant for plasma glucose disappearance; MSG, monosodium glutamate; PI, phosphatidylinositol; PI(3)K, phosphatidylinositol 3-kinase; PY, antiphosphotyrosine; SH, src homology.

Received May 9, 2003.

Accepted for publication September 24, 2003.

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