Reversal of denervation-induced insulin resistance by SHIP2 protein synthesis blockade

Daniela F. Bertelli1, Miriam Ueno2, Maria E. C. Amaral1, Marcos Hikari Toyama1, Everardo M. Carneiro1, Sergio Marangoni1, Carla R. O. Carvalho3, Mário J. A. Saad2, Lício A. Velloso2,*, and A. Carlos Boschero1,*

1 Departments of Physiology and Biophysics and 2 Internal Medicine, University of Campinas 6040 Campinas; and 3 Department of Physiology and Biophysics, University of São Paulo, 05508-900 Sao Paulo, Brazil


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Short-term muscle denervation is a reproducible model of tissue-specific insulin resistance. To investigate the molecular basis of insulin resistance in denervated muscle, the downstream signaling molecules of the insulin-signaling pathway were examined in intact and denervated soleus muscle of rats. Short-term denervation induced a significant fall in glucose clearance rates (62% of control, P < 0.05) as detected by euglycemic hyperinsulinemic clamp and was associated with a significant decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR; 73% of control, P < 0.05), IR substrate 1 (IRS1; 69% of control, P < 0.05), and IRS2 (82% of control, P < 0.05) and serine phosphorylation of Akt (39% of control, P < 0.05). Moreover, denervation reduced insulin-induced association between IRS1/IRS2 and p85/phosphatidylinositol (PI) 3-kinase. Nevertheless, denervation caused an increase in PI 3-kinase activity associated with IRS1 (275%, P < 0.05) and IRS2 (180%, P < 0.05), but the contents of phosphorylated PI detected by HPLC were significantly reduced in lipid fractions. In the face of the apparent discrepancy, we evaluated the expression and activity of the 5-inositol, lipid phosphatase SH2 domain-containing inositol phosphatase (SHIP2), and the serine phosphorylation of p85/PI 3-kinase. No major differences in SHIP2 expression were detected between intact and denervated muscle. However, serine phosphorylation of p85/PI 3-kinase was reduced in denervated muscle, whereas the blockade of SHIP2 expression by antisense oligonucleotide treatment led to partial restoration of phosphorylated PI contents and to improved glucose uptake. Thus modulation of the functional status of SHIP2 may be a major mechanism of insulin resistance induced by denervation.

denervation; SH2 domain-containing inositol phosphatase; phosphatidylinositol 3-kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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MUSCLE DENERVATION is a reproducible model of insulin resistance. It is characterized by a decreased ability of insulin to stimulate glucose uptake, glycogen synthesis, and amino acid transport (9). Several studies have attempted to characterize the major mechanisms involved in the development of impaired insulin action after denervation, and as it stands now, we know that modulation of muscle blood flow (31), reduced binding of insulin to its receptor (13), and loss of mechanical activity (10) are not responsible for the phenomenon. Defects in different steps of the insulin-signaling pathway are currently under scrutiny, and some advances have been achieved. No study was able to demonstrate a major loss of insulin receptor (IR) kinase activity (1, 20), although one study (14) demonstrated a reduced insulin-stimulated IR substrate 1 (IRS1) phosphorylation after 7 days of denervation. The activity of the lipid-metabolizing enzyme phosphatidylinositol (PI) 3-kinase was shown to be unaltered 30 min and 24 h after denervation and to decrease 3 days after denervation (2, 9). The activity of Akt was shown to be unaltered at 1 day and reduced at 3 days after denervation (30), and the ability of insulin to induce dephosphorylation of glycogen synthase was shown to be reduced 3 days after denervation (27). Finally, glucose transporters GLUT1 and GLUT4 undergo significant decrease in mRNA and protein expression 1 day after denervation, with no changes if analyzed after shorter periods (4). Thus a yet unclear picture suggests that defects at several levels and branches of the insulin-signaling cascade, occurring at different time points after denervation, participate in the mechanisms that produce the final phenotype of insulin resistance.

The objective of the present study was to evaluate the expression and functional activity of molecular elements participating in insulin signaling from IR to Akt in a model of skeletal muscle insulin resistance due to short-term denervation. Besides differences in insulin-induced tyrosine or serine phosphorylation in most elements analyzed, an apparent incongruent finding of increased IRS1- and IRS2-associated PI 3-kinase activity, paralleled by lower insulin-stimulated levels of phosphorylated PI in denervated muscle lipid extracts, led us to investigate the role of the recently described lipid phosphatase SH2 domain-containing inositol phosphatase (SHIP2). Although no changes in SHIP2 protein expression could be detected, a reversal of insulin resistance was achieved by treating denervated rats with an antisense oligonucleotide capable of blocking SHIP2 protein synthesis.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Materials. Reagents for SDS-PAGE and immunoblotting were from Bio-Rad (Richmond, CA). HEPES, phenylmethylsulfonyl fluoride (PMSF), antipain, aprotinin, leupeptin, pepstatin, benzamidine hydrochloride, dithiothreitol (DTT), ATP, PI 4-monophosphate, Triton X-100, Tween 20, glycerol, and bovine serum albumin (fraction V) were from Sigma (St. Louis, MO). Nonidet P-40 was from Calbiochem (La Jolla, CA), PI was from Avanti (Alabaster, AL), silica gel TLC plates were from Merck (Gibbstown, NJ), protein A-Sepharose 6 MB was from Pharmacia (Uppsala, Sweden), 125I-labeled protein A was from ICN Biomedicals (Costa Mesa, CA), [gamma -32P]ATP was from DuPont-New England Nuclear (Beverly, MA), and nitrocellulose paper (BA85, 0.2 µm) was from Amersham (Aylesbury, UK). Thiopental sodium (Amytal) and recombinant human insulin (Humulin R) were from Lilly (Indianapolis, IN). Polyclonal anti-phosphotyrosine antibodies were raised in rabbit and affinity-purified on phosphotyramine columns (19). Anti-IR (rabbit; sc-711), anti-IRS1 (rabbit; sc-559), anti-IRS2 (goat; sc-1555), anti-SHIP2 (goat; sc-14504), and anti-phospho[Ser473]Akt (goat; sc-1618) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rat-p85/PI 3-kinase (rabbit; no. 06-195) antiserum was from UBI (Lake Placid, NY). Anti-phospho-serine (rabbit; AB1603) antiserum was from Chemicon. The catheters were made of Tygon (Norton Performance Plastics, Akron, OH), polyvinyl (ICI Australia, Silverwater, BC, Australia), and polyethylene (PE 50; Clay Adams, Parsippany, NJ).

Experimental animals. Eight-week-old Wistar male rats (150-180 g) were allowed access to standard rodent chow and water ad libitum. Food was withdrawn 8 h before experiments. For each set of experiments, rats were submitted to surgical denervation of only one hindlimb, and soleus muscle specimens were collected from both the denervated and the intact limbs. The experimental protocol was approved by the University of Campinas Ethical Committee.

Denervation. The hindlimb was denervated as previously described (29). Rats were briefly anesthetized with thiopental sodium (15 mg/kg body wt ip) and used 10-15 min thereafter, i.e., as soon as anesthesia was assured by loss of pedal and corneal reflexes. A small superficial incision was made at the most proximal portion of the hindlimb, the thigh muscles were bluntly separated from the lateral side, the sciatic nerve was exposed, and ~0.5 cm of the nerve was removed. A sham operation was performed in the contralateral leg. Incisions on both legs were closed with surgical clips, and a topical disinfectant was applied to the skin.

Protein extraction, immunoprecipitation, and immunoblotting. Four hours after denervation, the rats were anesthetized again with thiopental sodium (15 mg/kg body wt ip), the abdominal cavity was opened, and 0.5 ml of saline (0.9% NaCl, with or without 10-6 M insulin) was injected in the cava vein. Ninety seconds after insulin injection, the soleus muscle was excised and homogenized. The extracts were centrifuged at 12,000 rpm at 4°C for 1 h to remove insoluble material, and equal amounts of the supernatant were used in immunoprecipitation experiments with anti-IR, anti-IRS1, or anti-IRS2 antibodies. The samples were processed for SDS-PAGE and Western blotting (6).

For protein analysis by immunoblotting, the samples were treated with Laemmli sample buffer containing 100 mmol/l DTT and heated in a boiling water bath for 4 min. For total extracts, 200-µg aliquots were subjected to SDS-PAGE (6% Tris-acrylamide) in a Bio-Rad miniature lab gel apparatus (Mini-Protean, Bio-Rad).

The nitrocellulose blots were incubated for 4 h at 22°C with anti-phosphotyrosine antibody, anti-IR antibody, anti-IRS1 antibody, anti-IRS2 antibody, anti-p85/PI3-kinase antibody, anti-phospho-serine antibody or anti-phospho[Ser473]Akt. The blots were incubated with 2 µCi of 125I-labeled Protein A (30 µCi/µg) in 10 ml of blocking buffer for 1 h at 22°C and washed again as described above for 2 h. 125I-Protein A bound to the antibodies was detected by autoradiography with preflashed Kodak XAR film (Eastman Kodak, Rochester, NY) with Cronex Lighting Plus intensifying screens (DuPont, Wilmington, DE) at -70°C for 12-48 h. Band intensities were quantified by densitometry (Scion software, ScionCorp) of the developed autoradiogram.

PI 3-kinase activity assay. PI 3-kinase activity was measured by in vitro phosphorylation of PI, as previously described with minor modifications (17, 21, 32). After insulin injection into the portal vein, a portion of the hindlimb skeletal muscle was removed and homogenized immediately in (1:10, wt/vol) ice-cold solubilization buffer (buffer B) with a Polytron PTA 20S generator (model PT 10/35, Brinkmann Instruments) operated at maximum speed for 30 s. The sample was then placed on ice for 30 min. The solubilization buffer B was composed of (in mmol/l) 50 HEPES (pH 7.5), 137 NaCl, 1 MgCl2, 1 CaCl2, 2 Na3VO4, 10 sodium pyrophosphate, 10 sodium fluoride, 2 EDTA, and 1% Nonidet P-40, 10% glycerol, aprotinin (2 µg/ml), antipain (10 µg/ml), leupeptin (5 µg/ml), pepstatin (0.5 µg/ml), benzamidine (1.5 µg/ml), and PMSF (34 µg/ml). Insoluble material was removed by centrifugation at 15,000 rpm in a Ti-70 rotor (Beckman) for 50 min. IRS1 and IRS2 were immunoprecipitated from aliquots of the supernatants containing 4 mg of total protein utilizing anti-IRS1 or anti-IRS2 antisera followed by protein A-Sepharose 6 MB. Alternatively, anti-rat p85 PI 3-kinase antibodies (whole serum,1 µl/ml) were used. The immunoprecipitates were washed successively in PBS containing 1% Nonidet P-40 and 100 mmol/l Na3VO4 (three times), 100 mmol/l Tris (pH 7.5) containing 500 mmol/l LiCl2 and 100 mmol/l Na3VO4 (three times), and 10 mmol/l Tris (pH 7.5) containing 100 mmol/l NaCl, 1 mmol/l EDTA, and 100 µM Na3VO4 (twice). The pellets were resuspended in 50 µl of 10 mmol/l Tris (pH 7.5) containing 100 mmol/l NaCl and 1 mmol/l EDTA. To each pellet were added 10 µl of 100 mmol/l MgCl2 and 10 µl of PI (2 µg/µl) sonicated in 10 mmol/l Tris (pH 7.5) with 1 mmol/l EGTA. The PI 3-kinase reaction was started by the addition of 10 µl of 440 µmol/l ATP containing 30 µCi of [32P]ATP. After 10 min at room temperature with constant shaking, the reaction was stopped by the addition of 20 µl of 8 N HCl and 160 µl of CHCl3-methanol (1:1). The samples were centrifuged, and the lower organic phase was removed and applied to a silica gel TLC plate (Merck) coated with 1% potassium oxalate. TLC plates were developed in CHCl3-CH3OH-H2O-NH4OH (60:47:11.3:2), dried, and visualized by autoradiography. The radioactivity in spots, which comigrated with a PI-4 standard, was measured by densitometry of the autoradiographic images obtained (Scion software).

Clamp studies. All procedures for clamp studies followed a previously published description of the method (7). After animals were fasted for 6 h, a 2-h hyperinsulinemic euglycemic clamp study was performed in the denervated or sham-operated limb. Under thiopental sodium anesthesia and aseptic conditions, a monoocclusive polyethylene catheter was inserted into the femoral artery for infusion of insulin and glucose. A second polyvinyl catheter was inserted into the femoral vein for blood sampling, and the animal was kept in a heated box (37°C) throughout the study. During the first phase of the study (30 min), a priming dose of insulin was infused followed by a rate of glucose infusion necessary to reach a plateau. After glucose equilibration, insulin infusion (3 mU · kg-2 · min-1) was maintained for 2 h at a constant rate (0.20 ml/h), and a variable infusion of glucose (5% solution) was adjusted to maintain the plasma glucose concentration at ~120 mg/dl. Blood samples were collected from the femoral vein every 5 min for plasma glucose and every 30 min for plasma insulin determinations. Insulin was measured in duplicate by radioimmunoassay and oscillated between 6.0 and 11.0 ng/ml in denervated limb blood samples and between 4.0 and 9.0 ng/ml in sham-operated limb blood samples.

HPLC analysis. The methods used were previously published (16, 26, 34). For identification of PIs, soleus muscle was excised and homogenized in 1.8 ml of a mixture of methanol-chloroform-HClO4 (8%) (20:10:1). After addition of 500 µl of chloroform and 500 µl of 1% HClO4, the lower organic phase was collected, washed twice with 1% HClO4, and evaporated. Deacylation was made as described previously (16). The product of the deacylation was resolved on an anion-exchange column (Shodex Aschipak, E8502 N7C) with the gradient of 980 µM (A), 3 M NaH2PO4 (B), pH 3.8. The linear gradient rose to 7% over 30 min, and a 1-min step to 15% was followed by a linear gradient to 30% B at 60 min, followed by a linear gradient to 60% B at 80 min. Finally, buffer B was increased to 100% over 5 min. Nonphosphorylated PI control was obtained by resolving highly purified PI from Avanti (which peaked at 25 min) in parallel with sample analysis. Phosphorylated PIs were detected as a peak occurring at 28-29 min.

Sense and antisense oligonucleotide studies. Sense and antisense oligonucleotides were diluted to a final concentration of 20 µmol/l in dilution buffer containing 10 mmol/l Tris · HCl and 1 mmol/l EDTA. The rats were injected (ip) with 200 µl of dilution buffer with or without sense or antisense oligonucleotides 2 h before and immediately after denervation. Phosphothioate-modified oligonucleotides were designed according to NM 022944 Rattus norvegicus SHIP-2 sequence and were composed of sense (5'-CTG CGG AGG AGC TGC T-3') and antisense (5'-AGC AGC TCC TCC GCA G-3') primers.

Statistical analysis. The results obtained in the denervated limb were always compared with data obtained in the sham-operated limb in experiments performed in parallel. Student's t-test for paired samples was used for comparison. The level of significance was set at P < 0.05.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Protein expression and insulin-induced activation/phosphorylation of elements participating in the insulin-signaling cascade. Binding of circulating insulin to its receptor promotes beta -subunit autophosphorylation and triggers a series of intracellular events responsible for the final effects of insulin in a given cell. In the present model of muscle insulin resistance, no changes in IR protein content were detected (Fig. 1A), but a significant fall in insulin-induced IR tyrosine phosphorylation was observed (Fig. 1B, 73% of control, P < 0.05). The protein contents of the main substrates of the IR (IRS1 and IRS2) were unaffected by short-term denervation (Figs. 2A and 3A). Nonetheless, insulin-induced tyrosine phosphorylation of either IRS1 (69%, P < 0.05) or IRS2 (82%, P < 0.05) was reduced (Figs. 2B and 3B). A metabolically active branch of the insulin-signaling cascade depends on activation of the lipid-metabolizing enzyme PI 3-kinase. To be activated by insulin stimulus, the p85 subunit of the PI 3-kinase must be engaged by either IRS1 or IRS2. In shortly denervated soleus muscle, the insulin-induced association of p85 with IRS1 (73%, P < 0.05) or IRS2 (73%, P < 0.05) was significantly reduced (Figs. 2C and 3C). However, the activity of the associated PI 3-kinase, as measured by its capacity to incorporate phosphorus in PI, was unexpectedly increased (275%, P < 0.05 for IRS1 association and 180%, P < 0.05 for IRS2 association; Figs. 2D and 3D). Divergence between IRS1- and IRS2-associated p85/PI 3-kinase amounts and the associated PI 3-kinase activity have been observed in other situations, and differences in the level of serine phosphorylation of p85/PI 3-kinase were mentioned as a possible reason for the observed phenomenon (11, 12, 32). Therefore, membranes from IRS1 and IRS2 immunoprecipitates were stripped and reblotted with anti-phospho-serine antibodies. As depicted in Fig. 4, a significantly lower level of serine phosphorylation is detected in the associated p85/PI 3-kinase (52 and 46% of control for IRS1 and IRS2, respectively, P < 0.05), which possibly explains the increased associated PI 3-kinase activity despite its lower association with the IRSs. The resulting PI3'4'P and PI3'4'5'P serve as docking sites for the enzymes Akt and phosphoinositide-dependent kinase 1 (PDK1). Apparently, PDK1 binds preferentially to PI3'4'P and, once in the same subcellular location of Akt (which binds with higher affinity to PI3'4'5'P), catalyzes its serine phosphorylation and activation. Despite the higher IRS1- and IRS2-associated PI 3-kinase activity, in recently denervated soleus muscle, a significantly lower level of serine-phosphorylated Akt was detected (39%, P < 0.05; Fig. 5). In the face of the incongruent finding of higher IRS1/IRS2-associated PI 3-kinase activity in a known model of insulin resistance, and occurring in parallel with reduced insulin-induced IR, IRS1, and IRS2 tyrosine phosphorylation and insulin-induced Akt serine phosphorylation, we decided to measure the amounts of phosphorylated species of PI in lipid extracts of insulin-stimulated intact (sham-operated) and denervated soleus muscle.


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Fig. 1.   Expression of insulin receptor (IR) and levels of tyrosine phosphorylation of IR in denervated and sham-denervated soleus muscle. Equal amounts of protein (4 mg), extracted from sham-denervated (Ctrl) and denervated muscles (Den), were subjected to immunoprecipitation (IP) with anti-IR antibody and immunoblotting (IB) with anti-IR antibody (A) or anti-phosphotyrosine (PY; B). Intensities of bands in autoradiograms were normalized to levels of insulin-stimulated sham-denervated muscles, noted as 100%. Results are means ± SE of 10 independent experiments (*P < 0.05, vs. Ctrl + insulin).



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Fig. 2.   Expression and levels of tyrosine phosphorylation of IR substrate 1 (IRS1), insulin-stimulated association of IRS1 with phosphatidylinositol (PI) 3-kinase, and IRS1-associated PI 3-kinase activity in denervated and sham-denervated soleus muscle. Equal amounts of protein (4 mg), extracted from sham-denervated (Ctrl) and denervated muscles (Den), were subjected to PI with IRS1 antibody (A-D) and IB with IRS1 antibody (A), PY (B), and with anti-p85 (C). The IRS1-associated PI 3-kinase activity is shown in D. Intensities of bands in autoradiograms were normalized to levels of insulin-stimulated sham-denervated muscles, noted as 100%. Results are means ± SE of 4-15 independent experiments (*P < 0.05, vs. Ctrl + insulin). PPI, phosphorylated PI.



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Fig. 3.   Expression and levels of tyrosine phosphorylation of IRS2, insulin-stimulated association of IRS2 with PI 3-kinase, and IRS2-associated PI 3-kinase activity in denervated and sham-denervated soleus muscles. Equal amounts of protein (4 mg), extracted from sham-denervated (Ctrl) and denervated muscles (Den), were subjected to PI with IRS2 antibody (A-D) and IB with IRS2 antibody (A), PY (B), and anti-p85 (C). IRS2-associated PI 3-kinase activity is shown in D. Intensities of bands in autoradiograms were normalized to levels of insulin-stimulated sham-denervated muscles, noted as 100%. Results are expressed as means ± SE of 4-12 independent experiments (*P < 0.05, vs. Ctrl + insulin).



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Fig. 4.   Serine phosphorylation of IRS1- and IRS2-associated p85. Membranes from experiments presented in Figs. 2 and 3 were stripped and reblotted with anti-phospho-serine (pSer) antibody. Bands appearing at 85 kDa, corresponding to IRS1- (A) or IRS2- (B)-associated p85-PI 3-kinase were scanned and densitometrically measured. Intensities of bands in autoradiograms were normalized to levels of insulin-stimulated sham-denervated muscles, noted as 100%. Results are expressed as means ± SE of 6 independent experiments (*P < 0.05 vs. Ctrl + insulin).



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Fig. 5.   Levels of Ser473 phosphorylation of Akt in denervated and sham-denervated soleus muscles. Equal amounts of protein (200 µg), extracted from sham-denervated (Ctrl) and denervated muscles (Den), were subjected to IB with [Ser473]pAkt antibodies. Results are expressed as means ± SE of 3 independent experiments (*P < 0.05, vs. Ctrl + insulin).

Glucose clearance and accumulation of phosphorylated PIs. Muscle denervation is known to produce insulin resistance. To investigate whether the present model was in accordance with the current literature, denervated and intact rats were submitted to a hyperinsulinemic euglycemic clamp. As expected, denervation produced a significant fall in the rate of glucose uptake under high insulin stimulus (62% of control, P < 0.05; Fig. 6). Akt is one of the major participants in the insulin-induced activation of GLUT4 trafficking from an intracellular to a membrane pool. As we have stated, the presence of PI3'4'P and most PI3'4'5'Ps is necessary for activation of Akt and thus for mediating insulin-induced glucose uptake. Therefore, we evaluated the amount of phosphorylated PIs in lipid extract from soleus muscles obtained from intact and denervated hindlimbs. By using an anion exchange column, the PIs of the membranes were resolved by HPLC, and a significant reduction in total amount of phosphorylated membrane inositols was detected (Fig. 7B). Because ~95% of membrane PIs are composed of the 5' phosphorylated forms of PIs, we suspected that a dysfunction of the 5'-position phosphoinositide phosphatase SHIP2 could participate in the phenomena observed in recently denervated muscle. Thus we measured the protein amounts and phosphorylation status of SHIP2.


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Fig. 6.   Glucose uptake as evaluated by euglycemic hyperinsulinemic clamp study. Two-hour study of euglycemic hyperinsulinemic clamp detected a 38% reduction of glucose uptake in denervated (Den) compared with sham-denervated (Ctrl) muscle (n = 6, *P < 0.05, vs. Ctrl + insulin).



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Fig. 7.   Measurement of phosphorylated phosphoinositols in denervated and sham-denervated soleus muscle by HPLC. After extraction and deacylation, phospholipids were resolved on an anion-exchange column, as described in MATERIALS AND METHODS. Identification of peaks was determined on basis of comparison with mobility of PI. The peak appearing at 25 min corresponds to nonphosphorylated PIs as determined by resolution of purified PIs from Avanti (C). PPIs peak at 28-29 min and are present in high amounts in sham-denervated muscle (A) but almost absent in denervated muscle samples (B, n = 3).

Expression and tyrosine phosphorylation of SHIP2. SHIP2 is an SH2 domain-containing 5'inositide phosphatase involved in the control of the insulin signal (3). Because a significant reduction in total phosphorylated PIs was detected in soleus muscle of hindlimb-denervated rats (Fig. 7B), we performed experiments to determine the protein amount and tyrosine phosphorylation status of SHIP2. No difference in SHIP2 protein content was detected when total extracts obtained from soleus muscle of control and denervated hindlimbs were compared (Fig. 8A, 1st and 2nd lanes). However, when insulin-induced tyrosine phosphorylation of SHIP2 was evaluated, a significant increase was detected in denervated hindlimb soleus muscle compared with control (Fig. 8B). Moreover, even in non-insulin-stimulated samples, a higher-than-control tyrosine phosphorylation of SHIP2 was found that matched the levels of tyrosine phosphorylation of SHIP2 present in insulin-stimulated control muscle (Fig. 8B).


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Fig. 8.   Expression and levels of tyrosine phosphorylation of SH2 domain-containing inositol phosphatase (SHIP2) in denervated and sham-denervated soleus muscle, and HPLC analysis of PIP derivatives associated with SHIP2 oligonucleotide treatment. Equal amounts of protein (200 µg for immunoblot and 4 mg for immunoprecipitation), extracted from sham-denervated (Ctrl) and denervated muscle (Den), were subjected to IB with anti-SHIP2 antibody (A) or PI with anti-phosphotyrosine (PY) and IB with anti-SHIP2 antibody (B). Results are expressed as means ± SE of 5 independent experiments (*P < 0.05, vs. Ctrl + insulin). In A: WO, not exposed to oligonucleotide; SS, exposed to sense SHIP2 oligonucleotide; AS, exposed to antisense SHIP2 oligonucleotide. For HPLC (C to H), tissues were extracted 4 h after denervation, and phospholipids were extracted, deacylated, and resolved on an anion-exchange column, as described in MATERIALS AND METHODS. Identity of peaks was determined on basis of comparison with mobility of PI. Sham-denervated treated with sense SHIP2 oligonucleotide (C), sham-denervated treated with antisense SHIP2 oligonucleotide (D), PI control (E), denervated treated with sense SHIP2 oligonucleotide (F), denervated treated with antisense SHIP2 oligonucleotide (G), and PI control (n = 3) (H).

Rate of glucose clearance and accumulation of phosphorylated PI in rats treated with SHIP2 antisense oligonucleotide. To test the hypothesis of involvement of SHIP2 in the mechanisms of denervation-induced insulin resistance, control and denervated rats were treated with two doses of 6 nmol phosphothioate sense or antisense oligonucleotides. Treatment with antisense oligonucleotide significantly reduced SHIP2 protein expression (Fig. 8A, 5th and 6th lanes). Antisense, but not sense, oligonucleotide partially restored the rate of insulin-stimulated glucose clearance as detected by hyperinsulinemic euglycemic clamp (Fig. 9) and promoted a partial recovery of total phosphorylated PI amounts as detected by HPLC (Fig. 8G).


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Fig. 9.   Glucose uptake as evaluated by euglycemic hyperinsulinemic clamp study in rats treated with antisense SHIP2 oligonucleotide. The 2-h study of the euglycemic hyperinsulinemic clamp performed in rats treated with antisense (AS) SHIP2 oligonucleotides revealed a significant improvement of glucose uptake compared with animals treated with no oligonucleotide (WO) or treated with sense SHIP2 oligonucleotide (SS) (n = 4, *P < 0.05, vs. Ctrl + insulin).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The negative modulation of different steps of the insulin-signaling pathway has been demonstrated in several clinical and experimental situations in which insulin resistance prevails (22). Thus reduction in protein expression or functional status of the IR, IRS1, IRS2, PI 3-kinase, and Akt is known to occur at various degrees and to display specific characteristics during aging, dexamethasone or epinephrine treatment, hypertension, and obesity, among other situations (23). The characterization of the molecular mechanisms of insulin signaling in such a diversity of situations, known to be coupled with insulin resistance, has contributed to reinforce the concepts of multifactoriality and complexity of type 2 diabetes and insulin resistance.

In the present series of experiments, it is demonstrated that short-term denervation leads to impaired glucose uptake strictly in the denervated portion of the body. As in the sham-operated limb, muscle from the denervated side presents no changes in the protein amount of IR, as well as no modulation in IRS1 and IRS2 protein expression. However, insulin-induced IR, IRS1, and IRS2 tyrosine phosphorylation and p85-IRS1/2 association are significantly reduced in denervated muscle. Up to these steps of the insulin-signaling cascade, the data herein presented are somewhat similar to those presented in a few recent studies (14). Notwithstanding, when analyzing the next step of the pathway, we were surprised by an apparently incongruent finding. Thus, although a significant reduction in insulin-induced IRS1 and IRS2 tyrosine phosphorylation was accompanied by reduced binding of p85/PI 3-kinase in soleus muscle from denervated limbs, a significant increase in insulin-induced IRS1- and IRS2-bound PI 3-kinase activity was detected. Finally, and in accordance with recent studies (33), a significant reduction of insulin-stimulated serine phosphorylation of Akt was detected in denervated soleus muscle. Divergence between catalytic activity and association of p85/PI 3-kinase with either IRS1 and IRS2 was previously observed in angiotensin II-induced activation of elements of the insulin-signaling pathway (11, 12, 32), which was accounted for by differential serine phosphorylation of p85/PI 3-kinase. In the present model of insulin resistance, a significant reduction of IRS1- and IRS2-associated p85/PI 3-kinase serine phosphorylation was detected in soleus muscle of denervated hindlimb, which might explain the increase in associated PI 3-kinase activity detected. Another possibility is that, under the extreme biochemical conditions employed for protein extraction during PI 3-kinase activity assay, an unspecific activation of the enzyme could have taken place.

PI 3-kinase has a pivotal role in the molecular linkage between early and late cellular events triggered by a number of hormones, growth factors, and cytokines (8). Active PI 3-kinase mediates the incorporation of phosphate at the 3' position in membrane-bound phosphoinositides generating PI 3,4-bisphosphate (PI3,4P2) and PI 3,4,5-trisphosphate (PI3,4,5P3). These phosphorylated PIs participate in the control of cellular mitogenesis, apoptosis, membrane trafficking, oncogenesis, and nutrient uptake and stocking. As a substrate of the insulin-signaling pathway, PI 3-kinase is activated downstream to IRS1 and IRS2 and induces the activation of the serine kinase Akt. The activation of Akt/PKB depends on its binding through a pleckstrin homology (PH) domain to PI3,4P2 and PI3,4,5P3 present in cellular membrane. Once bound to membrane PIs, Akt becomes available for the catalytic activity of phosphoinositide-dependent kinase 1 (PDK1), which mediates threonine phosphorylation, and for a yet unknown serine kinase necessary for full activation of Akt (5). Because recent studies have emphasized the predominant role of PI3,4,5P3 as target and mediator of Akt activation, and because 5' position-phosphorylated PIs correspond to >90% of the membrane's PIs, we measured by HPLC the total amount of PIs in the cell membranes of denervated and sham-operated limb muscle. As shown in Fig. 7, denervation led to substantial reduction in the amount of phosphorylated PIs in cell membrane. Thus, although an apparent mechanistic explanation for reduced insulin-induced serine phosphorylation of Akt in denervated muscle was found, a question about the outcome of an increased PI 3-kinase activity induced by insulin remained. Supposing that enough substrate for PI 3-kinase (PI4,5P2 and PI4P1) should be available, a rate of PI dephosphorylation that would surpass the rate of phosphate incorporation might explain the present findings. Two recently identified phosphatases involved in PI 3-kinase signaling are known to dephosphorylate membrane-bound phospho-PIs PTEN and SHIP2. PTEN, a phosphatase and tensin homolog deleted on chromosome 10, is the protein product of the PTEN tumor suppressor gene that is highly associated with development of glioblastomas and endometrial carcinomas when mutated or lost (25). PTEN is a 3'-position PI phosphatase that antagonizes the actions of PI 3-kinase by reducing the amount of membrane-bound PI3,4P2 and PI3,4,5P3. The loss of PTEN activity leads to increased Akt activation, whereas hyperexpression of PTEN reduces the amounts of serine-phosphorylated Akt inducing cell cycle arrest and increased rate of apoptosis (28). As stated above, the highest levels of membrane-bound PIs are accounted for by the 5'-position phosphorylated PIs. Therefore, as in the present model, when a drastic reduction in total phosphor-PIs in the membranes is detected, an increased activity of 5'-position phosphatases should become the primary candidate to be investigated. SHIP2 is a member of the inositol polyphosphatase 5-phosphatase family and a potent negative regulator of insulin signaling and insulin sensitivity in vivo. In response to stimulation by insulin, SHIP2 is closely linked to signaling events mediated by PI 3-kinase and Ras/mitogen-activated protein kinase. The loss of SHIP2 leads to increased sensitivity to insulin, associated with an increased recruitment of the GLUT4 glucose transporter and increased glycogen synthesis in skeletal muscle (3). In the present model, no difference in SHIP2 protein content was detected between control and denervated muscle when immunoblot analysis of total protein extracts was performed. However, when tyrosine phosphorylation status of the 5' phosphatase was evaluated, a significant difference was detected between control and denervated soleus muscle in both insulin-treated and non-insulin-treated rats. In fact, muscle from non-insulin-stimulated animals presented a SHIP2 tyrosine phosphorylation status similar to that detected in insulin-stimulated control muscle.

The role for tyrosine phosphorylation of SHIP2 relative to its phosphatase activity is a matter of controversy. In initial reports it was suggested that tyrosine phosphorylation would hamper SHIP1 phosphatase activity (18, 24). However, in recent studies, the regulation of SHIP2 tyrosine phosphorylation by insulin and other growth factors was demonstrated, suggesting that it participates in the positive control of its phosphatase activity (15, 28).

To investigate the participation of SHIP2 in denervation-induced insulin resistance, we designed phosphothioate-modified antisense oligonucleotides against SHIP2 mRNA. We treated the experimental animals with two doses of the compound, which resulted in 50% reduction of SHIP2 content in both control and denervated soleus muscle (Fig. 8A). Restrained SHIP2 expression led to significant increase in membrane-bound total phosphorylated PIs and coincided with a reversal of denervation-induced decrease in insulin-induced glucose uptake, as evaluated by hyperinsulinemic euglycemic clamp.

Site-specific insulin resistance generated by acute denervation is accompanied by a complex interplay of functional modulatory events that include several participants of the insulin signal transduction cascade. In the present study, it is demonstrated that significantly reduced amounts of phosphorylated PIs are present in the membrane of denervated muscle, which is accompanied by increased steady-state and insulin-induced tyrosine phosphorylation of SHIP2. The blockade of SHIP2 protein expression by antisense oligonucleotide treatment reversed the most remarkable clinical manifestation of muscle insulin resistance, i.e., insulin-induced glucose uptake. Therefore, we suggest that functional modulation of SHIP2 may participate in the genesis of denervation-induced insulin resistance, and approaches that hamper SHIP2 action may serve as therapeutic methods in diabetes mellitus and syndromes of insulin resistance.


    ACKNOWLEDGEMENTS

These studies were supported by grants from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), CNPq/Pronex (Coordenação Nacional de Pesquisa e Desenvolvimento).


    FOOTNOTES

* L. A. Velloso and A. C. Boschero contributed equally as advisors to the present study.

Address for reprint requests and other correspondence: A. C. Boschero, Departamento de Fisiologia e Biofisica, IB - UNICAMP, Campinas SP, Brazil (E-mail: boschero{at}unicamp.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.

First published November 26, 2002;10.1152/ajpendo.00345.2002

Received 5 August 2002; accepted in final form 21 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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