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. 2007 Nov 1;586(Pt 2):659–671. doi: 10.1113/jphysiol.2007.142414

Acute physical exercise reverses S-nitrosation of the insulin receptor, insulin receptor substrate 1 and protein kinase B/Akt in diet-induced obese Wistar rats

José R Pauli 1, Eduardo R Ropelle 1, Dennys E Cintra 1, Marco A Carvalho-Filho 1, Juliana C Moraes 1, Cláudio T De Souza 1, Lício A Velloso 1, José B C Carvalheira 1, Mario J A Saad 1
PMCID: PMC2375587  PMID: 17974582

Abstract

Early evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance. Here, we investigated whether this insulin resistance, mediated by S-nitrosation of proteins involved in early steps of the insulin signal transduction pathway, could be reversed by acute physical exercise. Rats on a high-fat diet were subjected to swimming for two 3 h-long bouts, separated by a 45 min rest period. Two or 16 h after the exercise protocol the rats were killed and proteins from the insulin signalling pathway were analysed by immunoprecipitation and immunoblotting. We demonstrated that a high-fat diet led to an increase in the iNOS protein level and S-nitrosation of insulin receptor β (IRβ), insulin receptor substrate 1 (IRS1) and Akt. Interestingly, an acute bout of exercise reduced iNOS expression and S-nitrosation of proteins involved in the early steps of insulin action, and improved insulin sensitivity in diet-induced obesity rats. Furthermore, administration of GSNO (NO donor) prevents this improvement in insulin action and the use of an inhibitor of iNOS (l-N6-(1-iminoethyl)lysine; l-NIL) simulates the effects of exercise on insulin action, insulin signalling and S-nitrosation of IRβ, IRS1 and Akt. In summary, a single bout of exercise reverses insulin sensitivity in diet-induced obese rats by improving the insulin signalling pathway, in parallel with a decrease in iNOS expression and in the S-nitrosation of IR/IRS1/Akt. The decrease in iNOS protein expression in the muscle of diet-induced obese rats after an acute bout of exercise was accompanied by an increase in AMP-activated protein kinase (AMPK) activity. These results provide new insights into the mechanism by which exercise restores insulin sensitivity.

Nitric oxide (NO) is a free radical and biological signalling molecule produced by the intracellular enzyme, NO synthase (Lane & Gross, 1999). The reactivity of NO towards molecular oxygen, thiols, transition metal centres, and other biological targets enables NO to act as an ubiquitous cell-signalling molecule with diverse physiological and pathophysiological roles (Gross & Wolin, 1995). In this regard, NO can react with cysteine residues in the presence of O2 to form S-nitrosothiol (Stamler et al. 1992, 1997), altering the activity of proteins including H-ras (Lander et al. 1995), the olfactory cyclic nucleotide-gated channel (Broillet & Firestein, 1996) and glyceraldehyde-3-phosphate dehydrogenase (Molina y Vedia et al. 1992). The reversible regulation of protein function by S-nitrosation has led to the proposal that S-nitrosothiols function as post-translational modifiers, analogous to those created by phosphorylation or acetylation (Stamler et al. 1997).

A previous study reported that inducible nitric oxide synthase (iNOS), a cytokine-inducible proinflamatory mediator in several pathological conditions, is overexpressed in the muscle and fat of genetic and dietary models of obesity and type 2 diabetes (Perreault & Marette, 2001), and that this expression is associated with insulin resistance. The iNOS knockout mice are protected from muscle insulin resistance by related diet-induced obesity (Perreault & Marette, 2001). We have recently demonstrated that the insulin resistance induced by increased iNOS in obesity may be related to S-nitrosation of insulin signalling proteins, insulin receptor (IR), insulin receptor substrate 1 (IRS1) and protein kinase B (Akt) (Carvalho-Filho et al. 2005).

It is well established that exercise training, even acutely, can improve insulin sensitivity in the muscle of obese rats (Betts et al. 1993; Bruce et al. 2001). However, the molecular mechanisms involved in this improvement in insulin signalling are not fully understood. Exercise training is also associated with enhanced AMP-activated protein kinase (AMPK) signalling. AMPK activation can modulate NO production through down-regulation of iNOS protein expression (Pilon et al. 2004). Treatment with AICAR, an AMPK agonist, improves glucose homeostasis and insulin sensitivity (Winder, 2000; Fiedler et al. 2001).

In the light of these previous data, we investigated whether the improvement in insulin signalling, associated with acute exercise, could be associated with the down-regulation of iNOS protein expression and reduced S-nitrosation of IRβ, IRS1 and Akt, and accompanied by AMPK activation.

Methods

Animals and diet

Male Wistar rats from the University of Campinas Central Animal Breeding Center were used in the experiments. All experiments were approved by the Ethics Committee of the State University of Campinas (UNICAMP).

The 4-week-old Wistar rats were divided into three groups, control rats (C) fed standard rodent chow (composition, see Table 1), obese rats, fed on an obesity-inducing diet for 3 months (DIO) (composition, see Table 1), and a third group, which also received an obesity-inducing diet, but was submitted to a single bout of exercise (DIO + EXE).

Table 1.

Components of rat diet and rat chow

Standard chow High fat diet
Ingredients g kg−1 kcal kg−1 g kg−1 kcal kg−1
Cornstarch (Q.S.P.) 397.5 1590 115.5 462
Casein 200 800 200 800
Sucrose 100 400 100 400
Dextrinated starch 132 528 132 528
Lard — — 312 2808
Soybean Oil 70 630 40 360
Cellulose 50 — 50 —
Mineral Mix 35 — 35 —
Vitamin Mix 10 — 10 —
l-Cystine 3 — 3 —
Choline 2.5 — 2.5 —
Total 1000 3948 1000 5358
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Exercise protocol

Rats were accustomed to swimming for 10 min for 2 days. The animals swam in groups of three in plastic barrels of 45 cm in diameter that were filled to a depth of 60 cm, for two 3 h-long bouts, separated by a 45 min rest period, and the water temperature was maintained at ∼34°C. This exercise protocol was adaptated from a previously published procedure (Chibalin et al. 2000). Two and 16 h after the exercise protocol, or as indicated in the time course experiments, the rats were anaesthetized with an intraperitoneal (i.p.) injection of sodium thiopental (40 mg (kg body weight)−1). In all experiments the appropriateness of anaesthesia depth was tested by evaluating pedal and corneal reflexes, throughout the experimental procedure. Following the experimental procedures, the rats were killed under anaesthesia (thiopental 200 mg kg−1) following the recommendations of the NIH publication n°85–23.

S-Nitrosoglutathione treatment

S-Nitrosoglutathione (GSNO) was prepared by the reaction of glutathione with sodium nitrite in acidic solution, as previously reported (Shishido et al. 2003). The rats received an intraperitoneal (i.p.) injection of GSNO (0.1 mol l−1) or phosphate-buffered saline every 2 h, until completing four doses in 8 h. Immediately after the last dose the animals were submitted to the same exercise protocol, and 2 h or 16 h after the last bout of exercise, the rats were anaesthetized with an i.p. injection of sodium thiopental (40 mg (kg body weight)−1), and then skeletal muscle (gastrocnemius) was taken for biochemical analyses.

l-NIL treatment

The rats received an intraperitoneal injection of the iNOS inhibitor l-N6-(l-iminoethyl)lysine (l-NIL; 80 mg (kg body weight)−1) or phosphate-buffered saline twice (every 12 h) daily for 10 days. This treatment protocol with l-NIL was adaptated from a previously published procedure (Sugita et al. 2005). One hour after the last dose of l-NIL, the animals were submitted to the protocol of acute exercise previously described, and 2 h and 16 h after the last bout of exercise, the rats were anaesthetized with an i.p. injection of sodium thiopental (40 mg (kg body weight)−1), and then skeletal muscle (gastrocnemius) was extracted for biochemical analyses.

Insulin tolerance test (ITT) and serum insulin determination

Two hours and 16 h after the exercise protocol, the rats were submitted to an insulin tolerance test (ITT; 9.0 ml kg−1 of a solution 10−6 mol l−1 of insulin). Briefly, 9.0 ml kg−1 of a solution 10−6 mol l−1 of human recombinant insulin (Humulin R) from Eli Lilly (Indianapolis, IN, USA) was injected i.p. in anaesthetized rats, the blood samples were collected from the tail at 0, 5, 10, 15, 20, 25 and 30 min, for serum glucose determination. The rate constant for plasma glucose disappearance (KITT) was calculated using the formula 0.693/(t1/2). The plasma glucose t1/2 was calculated from the slope of least square analysis of the plasma glucose concentration during the linear phase of decline (Bonora et al. 1989). Plasma glucose level was determined by colorimetric method using a glucosemeter (Advantage. Boehringer Mannheim, USA). Plasma was separated by centrifugation (1100 g) for 15 min at 4°C and stored at −80°C until assay. Radioimmunoassay (RIA) was employed to measure serum insulin, according to a previous description (Scott et al. 1990).

Protein analysis by immunoblotting

As soon as anaesthesia was assured by the loss of pedal and corneal reflexes, the abdominal cavity was opened, the portal vein was exposed and 0.2 ml of normal saline with or without insulin (10−6 mol l−1) was injected. In preliminary experiments, we determined that this dose of insulin can reach peripheral levels that are 3–4 times higher than the dose that can induce the maximal insulin effect on insulin signalling proteins in muscle. At 90 s after the insulin injection, both portions of gastrocnemius (red and white fibres) were ablated, pooled, 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 PMSF and 0.1 mg ml−1 aprotinin) at 4°C with a Polytron PTA 20S generator (Brinkmann Instruments model PT 10/35) operated at maximum speed for 30 s. The extracts were centrifuged at 9000 g and 4°C in a Beckman 70.1 Ti rotor (Palo Alto, CA, USA) for 40 min to remove insoluble material, and the supernatants of these homogenates were used for protein quantification, performed by the Bradford method. Proteins were denatured by boiling in Laemmli sample buffer containing 100 mm DTT, run on SDS-PAGE, transferred to nitrocellulose membranes, which were blocked, probed and developed as previously described (Laemmli, 1970; Saad et al. 1997). The IRβ and IRS1 were immunoprecipitated from rat muscle with or without previous insulin infusion in the portal vein. Antibodies used for immunoblotting were antiphosphotyrosine (pY) (sc-508, mouse monoclonal) anti-IR (sc-711, rabbit polyclonal), anti-IRS1 (sc-559, rabbit polyclonal), anti-Akt (sc-1618, goat polyclonal), antiphospho [Ser473] Akt (sc-7985-R, rabbit polyclonal), anti-iNOS (sc-7271, mouse monoclonal), antiphospho-c-jun N-terminal kinase (JNK) (sc-6254, mouse polyclonal), anti-protein tyrosine phosphate 1B (PTP1B) (sc-1719 goat polyclonal) (Santa Cruz Biotechnology, CA, USA), antiphospho [Ser79] acetyl CoA carboxylase (ACC) (rabbit polyclonal, 07-184), antiphosphoserine-IRS-1307 (rabbit polyclonal, no 7247) was from Upstate Biotechnology (Charlottesville, VA, USA). Anti-ACC (rabbit polyclonal, no 3662), anti-AMPK (rabbit polyclonal, no 2757), antiphospho [Thr172] AMPK (rabbit polyclonal, no. 2531), antip85-PI3-kinase (phosphatidylinositol 3-kinase, PI3-K) (rabbit polyclonal, no 4292) antibodies were from Cell Signalling Technology (Beverly, MA, USA). Blots were exposed to preflashed Kodak XAR film with Cronex Lightning Plus intensifying screens at 80°C for 12–48 h. Band intensities were quantified by optical desitometry (Scion Image software, ScionCorp, Frederick, MD, USA) of the developed autoradiographs.

Detection of S-nitrosated proteins by the biotin-switch method

The biotin-switch assay was performed essentially as previously described (Jaffrey & Snyder, 2001; Martinez-Ruiz & Lamas, 2004). Muscle tissue was extracted and homogenized in extraction buffer (250 mm Hepes, pH 7.7, 1 mm EDTA, 0.1 mm neucuproine). After centrifugation at 9000 g for 20 min, insoluble material was removed and extracts were adjusted to 0.5 mg ml−1 of protein, and equal amounts were blocked with four volumes of blocking buffer (225 mm Hepes, pH 7.7, 0.9 mm neucuproine, 2.5% SDS, and 20 mm methylmethanethiosulphonate) at 50°C for 30 min with agitation. After blocking, extracts were precipitated with two volumes of cold acetone (−20°C), chilled at −20°C for 10 min, centrifuged at 2000 g at 4°C for 5 min, washed with acetone, dried out, and resuspended in 0.1 ml HENS buffer (250 mm Hepes, pH 7.7, 1 mm EDTA, 0.1 mm neucoproine, and 1% SDS) per milligram of protein. Until this point, all operations were carried out in the dark. A one-third volume of biotin-HPDP 4 mm and 2.5 mm ascorbic acid was added and incubated for 1 h at room temperature. Proteins were acetone-precipitated again and resuspended in the same volume of HENS buffer.

For purification of biotinylated proteins, samples from the biotin-switch assay were diluted with two volumes of neutralization buffer (20 mm Hepes, pH 7.7, 100 mm NaCl, 1 mm EDTA, and 0.5% Triton X-100), and 15 μl neutravidin-agararose per milligram of protein in the initial extract was added and incubated for 1 h at room temperature with agitation. Beads were washed five times with washing buffer (20 mm Hepes, pH 7.7, 600 mm NaCl, 1 mm EDTA, and 0.5% Triton X-100) and incubated with elution buffer (20 mm Hepes, pH 7.7, 100 mm NaCl, 1 mm EDTA, and 100 mm 2-mercaptoethanol) for 20 min at 37°C with gentle stirring. Supernatants were collected, Laemmli buffer was added, and proteins were separated by SDS-PAGE.

Other assays

Serum free fatty acids (FFA) levels were analysed in rats using the NEFA-kit-U (Wako Chemical GmBH, Neuss, Germany) with oleic acid as a standard. Serum levels of tumour necrosis factor α (TNF-α) was determined in rats using ELISA kits from Pierce Biotechnology (Rockford, IL, USA), according to the instructions of the manufacturer. Both analyses were determined 2 h after the acute exercise.

Statistical analysis

Where appropriate, the results were expressed as means ± s.e.m. Differences between the lean group and sedentary obese group and between the sedentary obese and the group submitted to the exercise protocol were evaluated using one-way analysis of variance (ANOVA). When ANOVA indicated significance, a Bonferroni post hoc test was performed.

Results

Physiological and metabolic parameters

Table 2 shows comparative data regarding controls (C), diet-induced obesity rats (DIO) and DIO rats submitted to exercise protocol (DIO + EXE-2 h and DIO + EXE-16 h). Rats fed on the high-fat diet for 12 weeks had a higher body weight, epididymal fat and fasting serum insulin than age-matched controls (C). No significant variations were found in body weight, epididymal fat and fasting serum insulin in DIO rats after a single session of exercise, compared to DIO rats. The fasting glucose concentrations were similar between the groups; however, the reduction in the glucose disappearance rate (KITT), induced by the high-fat diet, was restored 2 h and 16 h after acute exercise.

Table 2.

Characteristics of Wistar rats after 3 months on a high-fat diet (DIO), DIO rats submitted to exercise (DIO + EXE-2 h and DIO + EXE-16 h) and their age-matched controls

Control DIO DIO + EXE-2 h DIO + EXE-16 h
Body weight (g) 412.3 ± 17.4 536.8 ± 28.8* 539.1 ± 29.6* 575,1 ± 26,5*
Epididymal fat (g) 5.67 ± 1.2 11.34 ± 1.6* 11.41 ± 1.4* 12,63 ± 1.3*
Plasma glucose (mg dl−1) 81.80 ± 5.9 89.70 ± 7.1 90.63 ± 8.5 85,63 ± 9,6*
Insulin (ng ml−1) 3.33 ± 0.8 6.84 ± 0.66* 6.12 ± 1.8* 6.41 ± 1,8*
FFA (mmol l−1) 0.63 ± 0.16 1.72 ± 0.27* 2.92 ± 0.4*# ND
TNFα (pg ml−1) 42.6 ± 19.8 122.6 ± 14.1* 129.7 ± 17.4* ND
Kitt (% min−1) 4.67 ± 0,14 2.4 ± 0,6* 4.89 ± 0,8# 4.72 ± 0.6#
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n = 8 in each group.

*

P < 0.001 versus control group

#

P < 0.001 versus DIO #P < 0.001 versus DIO, ND, not determined.

As expected, the plasma FFA levels were higher in obese rats, and acute exercise induced a marked increase in the levels of this substrate. Plasma levels of TNF-α were also higher in DIO rats, but there was not a clear increase in this cytokine after a bout of exercise.

A single bout of exercise improves insulin signalling in the muscle of DIO rats

The effect of in vivoi.v. insulin infusion on IR tyrosine phosphorylation was examined in the gastrocnemius muscle of controls, DIO rats and DIO rats submitted to exercise (after 2 h and 16 h). Fragments of muscle tissue were immunoprecipitated with anti-IR antibody and then blotted with antiphosphotyrosine antibody. In the control animals, insulin increased IR tyrosine phosphorylation by 6.9-fold over basal, compared with 2.4-fold over basal in the muscle of DIO rats. Insulin increased IR tyrosine phosphorylation by 7.6-fold and 4.8-fold over basal in the muscle from DIO + EXE-2 h and DIO + EXE-16 h rats, respectively, showing that exercise improves insulin-induced IR tyrosine phosphorylation in these rats (Fig. 1A). There was no difference in basal levels of IR tyrosine phosphorylation or in IR protein levels between the groups (Fig. 1A).

Figure 1. Insulin signalling in the muscle of controls, DIO and DIO rats submitted to exercise.

Figure 1

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Muscle extracts from rats injected with saline or insulin were prepared as described in Methods. A, tissue extracts were immunoprecipitated (IP) with anti-IRβ antibody and immunoblotted (IB) with anti-PY antibody or anti-IRβ antibody. B, tissue extracts were also IP with anti-IRS1 antibody and IB with anti-PY antibody and anti-IRS1 antibody, or C, anti-PI3-K antibodies. D, muscle extracts were IB with anti-Akt or antiphospho-[Ser473] Akt antibodies. The results of scanning densitometry are expressed as arbitrary units. Bars represent means ± s.e.m. of eight rats. *P < 0.05, versus DIO rats.

IRS1 tyrosine phosphorylation and IRS1/PI3–K association were observed to increase in control animals by 8.0-fold and 8.6-fold over basal following insulin administration, respectively, compared with 2.4-fold and 1.9-fold increases in the muscle of DIO rats over basal. Insulin increased IRS1 tyrosine phosphorylation and IRS1/PI3–K association by 7.1-fold and 7.5-fold and 5.3-fold and 5.8-fold in the muscle from DIO + EXE-2 h and DIO + EXE-16 h rats, respectively, over basal, showing that exercise improves insulin-induced IRS1 tyrosine phosphorylation in these rats (Fig. 1B and C). There was no difference in basal levels of IRS1 tyrosine phosphorylation or in IRS1 protein levels between the groups (Fig. 1B).

Finally, in gastrocnemius muscle from control rats, insulin increased Akt serine phosphorylation by 7.7-fold over basal, compared with a 2.4-fold increase in the muscle from DIO rats over basal. Similar to insulin-induced IR and IRS1 tyrosine phosphorylation, there was an increase of 7.9-fold and 5.2-fold in the muscle of DIO + EXE-2 h and DIO + EXE-16 h rats, respectively, over basal, showing that exercise improves insulin-induced Akt phosphorylation in these rats (Fig. 1D). There were no differences between the basal levels of Akt serine phosphorylation or in Akt protein levels between the groups (Fig. 1D).

Acute physical exercise reverses S-nitrosation and restores insulin signalling in the muscle of diet-induced obesity rats

In order to determine the time-course of the exercise-induced reduction in iNOS expression, we measured iNOS protein levels by immunoblotting in the muscle of obese rats that were submitted to the exercise protocol at 1, 2, 4 and 16 h after exercise. Results show that 2 h after exercise there is greater decrease in iNOS protein expression and that after 16 h this expression is still decreased compared to controls (Fig. 2A). In the muscle of DIO rats an enhanced expression of iNOS was found (Fig. 2B). We demonstrated an enhanced S-nitrosation of IRβ, IRS1, and Akt in the muscle of DIO rats, and this nitrosation was significantly reduced 2 h and 16 h after exercise in DIO rats (Fig. 2C–E).

Figure 2. The protein iNOS expression and S-nitrosation of the proteins of the insulin signalling pathway in the gastrocnemius muscle of controls, DIO and DIO rats submitted to exercise.

Figure 2

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A, iNOS expression decreased 2 h after acute exercise, as demonstrated in the time course. However, the iNOS expression remained decreased for 16 h as compared to the DIO rats. B, iNOS expression was assessed by iNOS protein levels using Western blot, as described in Methods. C–E, S-nitrosation of IRβ (C), IRS1 (D), and Akt (E) is shown, as determined by the biotin-switch method. Bars represent means ± s.e.m. of eight rats. *P < 0.05, versus control and #P < 0.05, DIO + EXE versus DIO.

The increase in AMPK phosphorylation induced by acute physical exercise inhibits iNOS and reverses insulin resistance in the muscle

In order to investigate the time-course of the exercise-induced increase in AMPK phosphorylation, we measured p-AMPK levels by immunoblotting. Physical exercise stimulated AMPK phosphorylation, which peaked at 1 h and then decreased, but phosphorylation at 16 h was still higher than basal (Fig. 3A). Two hours after exercise, in obese animals, in parallel with an increase in AMPK phosphorylation there was also an important increase in ACC phosphorylation, a downstream target of AMPK and a good correlate of its activation (Winder 2000). However, in obese animals (DIO) there was a decreased in AMPK and ACC phosphorylation when compared with control and DIO rats submitted to acute exercise protocol (Fig. 3B and C).

Figure 3. Expression and phosphorylation of AMPK and ACC in rat gastrocnemius muscles.

Figure 3

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A, physical exercise stimulated AMPK phosphorylation which peaked at 1 h and then decreased, but at 16 h it was still higher than basal. B and C, the results show that, in obese animals, 2 h after exercise, in parallel to an increase in AMPK phosphorylation there was also a significant important increase in ACC phosphorylation, a downstream target of AMPK and a good correlate of its activation (Winder 2000). Bars represent means ± s.e.m. of 16 rats (C and DIO) and 8 rats (DIO + EXE) *P < 0.05, versus control and #P < 0.05, DIO + EXE versus DIO.

GSNO induces insulin resistance in muscle gastrocnemius in vivo by means of S-nitrosation

In order to investigate whether supplementation with a NO donor could abolish the effect of exercise in DIO rats, we administrated GSNO to obese rats for 8 h (every 2 h), and these animals were then submitted to the exercise protocol. Results showed that, in animals treated with GSNO, the effect of exercise on insulin sensitivity was abolished, as indicated by a lower plasma glucose disappearance rate during the insulin tolerance test (KITT) (Fig. 4A), suggesting that the reduction in nitrosation may be an important mechanism for the improvement of insulin sensitivity induced by exercise. In accordance with these data, insulin-induced IR, IRS1 and Akt phosphorylation did not increase in GSNO-treated animals after exercise (Fig. 4B–D).

Figure 4. Effect of GSNO on insulin sensitivity and S-nitrosation in muscle of controls, DIO and DIO rats submitted to exercise.

Figure 4

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A, effect of GSNO treatment on glucose disappearance rates, measured by the 30-min insulin tolerance test (KITT). B–D, insulin-induced tyrosine phosphorylation of IRβ (B), IRS1 (C) and serine phosphorylation of Akt (D) followed by immunoprecipitation are shown. E, iNOS protein expression assessed by immunoblot is indicated. F–H, S-nitrosation of IRβ (F), IRS1 (G) and Akt (H) in muscle of animals, determined by the biotin switch method are shown. Bars represent means ± s.e.m. of six rats. *P < 0.05, versus control; #P < 0.05, DIO + EXE versus DIO; ##P < 0.05, versus DIO or DIO + EXE-2 h plus GSNO injection.

In the muscle of DIO rats, we found an enhanced expression of iNOS, and acute exercise reduces iNOS expression in GSNO-treated rats. (Fig. 4E). As expected, nitrosation of IRβ, IRS1, and Akt was not reduced after exercise in GSNO-treated rats (Fig. 4F–H). These data suggest that the maintenance of nitrosation of IR, IRS1 and Akt by GSNO precludes the effect of exercise on insulin sensitivity.

The effects of an iNOS inhibitor on insulin resistance and S-nitrosation of proteins of the insulin signalling pathway

We next investigated whether an inhibitor of iNOS (l-NIL) could mimic the effect of exercise on insulin sensitivity and signalling in DIO rats. The administration of l-NIL for 10 days improved insulin sensitivity in DIO rats, as measured by the glucose disappearance rate during the insulin tolerance test, KITT, and no additive effect was observed in DIO animals that were treated with this drug and that exercised (Fig. 5A). In these experiments, insulin-induced IR, IRS1 and Akt phosphorylation was improved by l-NIL, furthermore, no additive effect was observed with exercise (Fig. 5B–D). l-NIL inhibits iNOS activity, but our results show that l-NIL has no effect on iNOS protein expression. As expected, l-NIL reduced IR, IRS1 and Akt S-nitrosation in DIO rats, and again no additive effect was observed with exercise (Fig. 5E–H).

Figure 5. Effect of iNOS inhibitor (l-NIL) on insulin sensitivity and S-nitrosation in muscle of controls, DIO and DIO rats submitted to exercise.

Figure 5

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A, effect of treatment for 10 days with l-NIL on glucose disappearance rates, measured by the 30-min insulin tolerance test (KITT). B–D, insulin-induced tyrosine phosphorylation of IRβ (B), IRS1 (C) and serine phosphorylation of Akt (D) followed by immunoprecipitation are shown. E, iNOS protein expression assessed by immunoblot is indicated. F–H, S-nitrosation of IRβ (F), IRS1 (G) and Akt (H) in muscle of animals, determined by the biotin switch method are shown. Bars represent means ± s.e.m. of six rats. *P < 0.05, versus control and #P < 0.05, DIO + EXE versus DIO without GSNO injection.

Effect of treatment with GSNO or l-NIL on PTP1B protein levels, JNK activity and IRS1 serine phosphorylation in the muscle of controls, DIO and DIO + EXE rats

Diet-induced obesity is associated with an increased expression of PTP1B in muscle, and 2 h after an acute bout of exercise there is no change in this protein expression (Fig. 6A). The previous administration of a NO donor, GSNO, did not modify this increased expression of PTP1B.

Figure 6. Effect of GSNO or l-NIL on PTP1B protein levels, JNK activity and IRS1 serine phosphorylation in the muscle of controls, DIO and DIO rats submitted to exercise.

Figure 6

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Tissue extracts were immunoblotted (IB) with anti-PTP1B antibody (A and D), antiphospho-JNK antibody (B and E), anti-IRS1307 phosphoserine antibody (C and F). Bars represent means ± s.e.m. of eight rats. *P < 0.05, versus control.

JNK activation was determined by monitoring phosphorylation of JNK (Thr 183 and Tyr 185) (Fig. 6B). The high-fat diet induced an increase in JNK phosphorylation in the muscle of DIO rats when compared with control rats. Two hours after the exercise protocol is not sufficient to reverse the increase in JNK phosphorylation in the muscle of DIO rats. This phenomenon is not affected by a previous administration of GSNO. Similar results were observed for IRS1 Ser307 phosphorylation (Fig. 6C).

The administration of l-NIL, which is an inhibitor of iNOS, did not affect the increase in PTP1B protein expression (Fig. 6D), JNK phosphorylation (Fig. 6E), or IRS1 Ser307 phosphorylation (Fig. 6F), induced by high-fat diet.

Discussion

The impaired insulin action on whole-body glucose uptake is a hallmark feature of type 2 diabetes mellitus. Physical exercise has been linked to improved glucose homeostasis and enhanced insulin sensitivity after an acute bout of exercise in humans (Devlin et al. 1987; Zierath, 1995) and rodents (Richter et al. 1982; Wallberg-Henriksson, 1987; Wallberg-Henriksson et al. 1988). In this study, we demonstrated that a high-fat diet leads to an increase in the iNOS protein level and S-nitrosation of IRβ, IRS1 and Akt. Interestingly, an acute bout of exercise reduces iNOS expression and S-nitrosation of proteins involved in the early steps of insulin action and improves insulin sensitivity in DIO rats.

Several mechanisms may be involved in insulin resistance in the muscle of DIO rats, including serine phosphorylation of IR or IRSs (induced by serine kinases such as mTOR, and the stress kinases, JNK and IKKβ IAB kinase), by an increase in the activity or amount of the enzymes that normally reverse insulin action (e.g. PTP1B) or by an increase in iNOS (Hotamisligil et al. 1996; Bedard et al. 1997; Elchebly et al. 1999; Perreault & Marette, 2001; Hirosumi et al. 2002; Carvalho-Filho et al. 2005; Ropelle et al. 2006). In several situations of insulin resistance, such as diet-induced or genetic obesity, and endotoxaemia, iNOS is induced in tissues classically related to insulin signalling (Bedard et al. 1997; Kapur et al. 1997; Kapur et al. 1999). Perreault & Marette (2001) demonstrated that the genetic disruption of iNOS protects against obesity-linked insulin resistance, preventing impairments in PI3-K and Akt activation by insulin in muscle.

We recently demonstrated that the insulin resistance associated with iNOS induction is mediated by S-nitrosation of proteins involved in insulin signal transduction, i.e, insulin receptor β-subunit, insulin receptor substrate 1 (IRS1), and Akt. S-nitrosation of IRβ reduces its autophosphorylation and tyrosine-kinase activity and S-nitrosation of IRS1 is associated with its reduced tissue expression (Carvalho-Filho et al. 2005; Carvalho-Filho et al. 2006). In addition, S-nitrosation of Akt is associated with a decreased serine kinase activity of this enzyme in basal states and after insulin stimulation. S-nitrosation of these proteins is associated with the down-regulation of the IRβ/IRS1/PI3-K/Akt pathway. Since this pathway plays a central role in the metabolic actions of insulin in the muscle, including stimulation of glucose uptake and glycogen synthesis, down-regulation of this pathway in muscle by S-nitrosation may be an important mechanism of iNOS-induced insulin resistance. Interestingly, our data show that a single bout of exercise in DIO rats reduced iNOS expression and the S-nitrosation of IRβ/IRS1/Akt in skeletal muscle. In accordance, it has recently been demonstrated in muscle biopsies that a regular exercise programme reduces the local expression of cytokines and iNOS (Gielen et al. 2003). Our data show that the effect of exercise, in reducing iNOS expression and decreasing S-nitrosation of proteins involved in early steps of insulin action, was accompanied by an improvement in insulin sensitivity and an increase in insulin-induced IRβ, IRS1 and Akt phosphorylation. In addition, our data reinforce this mechanism by showing, first, that treatment with a NO donor (GSNO) prevents the beneficial effect of exercise on insulin sensitivity and, secondly, by showing that upon inhibition of this latter situation there is no additive effect of exercise.

Some of our results differ from other studies on acute exercise (Chibalin et al. 2000; Arias et al. 2007), which showed unchanged serine phosphorylated Akt, but increased threonine phosphorylated Akt. The reasons for these differences are not completely clear, but certainly methodological differences may contribute to these discrepancies. In previous reports (Chibalin et al. 2000; Arias et al. 2007), insulin signalling was investigated in isolated muscle after in vitro incubation; however, our results were obtained after in vivo insulin stimulation. In addition, these previous studies investigated control rats, while in the present study we investigated the effect of exercise on DIO rats.

Exercise and muscle contraction have been shown to enhance NO production and NOS expression in muscle (Balon & Nadler, 1997). Previous data show that low doses of NO donors can increase glucose uptake and improve insulin action (McGrowder et al. 2006). We also investigated whether administration of low doses of GSNO (1 μm) can induce improved glucose uptake and/or changes in S-nitrosation of proteins during the early steps of insulin action in the muscle of rats. These results showed that at low doses, an NO donor (GSNO) improved glucose uptake, but did not induce S-nitrosation of IR, IRS1, PI3-K or Akt in muscle (data not shown). Although the mechanisms by which administration of low doses of NO can improve insulin sensitivity are not well established, our data suggest that changes in the S-nitrosation status of proteins involved in the insulin signalling pathway are not involved. In addition, it is important to emphasize that exercise induces neuronal and endothelial nitric-oxide synthase (nNOS and eNOS, also termed NOS1 and NOS3, respectively) and not iNOS (also termed NOS2) expression. We can, thus, suggest that the effect of NO on insulin sensitivity is dose dependent and also depends on the enzyme that generates NO, which is expressed in different sites.

The mechanism by which exercise reduces iNOS expression in DIO rats is not completely understood, but it may be mediated, at least in part, by an increase in AMPK. It is well known that exercise activates AMPK, and this is believed to contribute to its insulin-sensitizing action in situations of insulin resistance (Wojtaszewski et al. 2005). Data from different sources indicate that AMPK activation reduces iNOS induction and blunts iNOS-mediated NO production (Bedard et al. 1997; Kapur et al. 1997; Pilon et al. 2004). In accordance with these previous data, we show that, in the muscle of DIO rats, an acute bout of exercise is accompanied by an increase in AMPK activation and a reduction in iNOS expression.

In addition, it should be taken into consideration that insulin resistance is a metabolic situation that is related to several molecular mechanisms that act in parallel to down-regulate insulin signalling. Since exercise is an efficient way to improve insulin sensitivity, it is possible that it may also act in other mechanisms of insulin resistance. In this regard, we recently verified an attenuation of the increase in the expression and activity of the PTP1B 16 h after a single session of exercise (Ropelle et al. 2006). In this same exercise protocol, we also observed the reverse of JNK activation and the increase of serine phosphorylation of IRS1 in muscle of DIO rats.

Taking these data together with our data, we can suggest that exercise reverses the multiple mechanisms that can contribute to insulin resistance. However, the present data show that GSNO, which induces insulin resistance and impairs exercise-induced improvement in insulin action, does not affect JNK phosphorylation, PTP1B expression or IRS1 serine phosphorylation. In addition l-NIL, an inhibitor of iNOS improves insulin action and insulin signalling in DIO rats without influencing JNK phosphorylation, PTP1B expression or IRS1 serine phosphorylation. These data suggest that S-nitrosation is an important mechanism of insulin resistance, and reversal of this mechanism is sufficient to improve insulin action and signalling.

It is possible that common upstream events could mediate the increased activation of JNK, S-nitrosation and iNOS expression in DIO rats, and that this common mechanism is reversed by exercise. We investigated some candidates and our data showed that 2 h after exercise there is a marked increase in FFA levels and almost no change in TNF-α levels, suggesting that these are probably not common upstream events in DIO and are not reversed by exercise. It is important to emphasize that JNK, S-nitrosation and iNOS can be activated by toll-like receptor 4 (TLR4), which is a pattern recognition receptor also present in muscle (Shi et al. 2006; Tsukumo et al. 2007). It was recently demonstrated in humans that exercise down-regulates TLR4 in monocytes (Lancaster et al. 2005), suggesting that this receptor may be a candidate for the activation of JNK, iNOS and S-nitrosation in obesity, and may be reversed by exercise.

It has been recently demonstrated that in humans, 15–18 h after a single session of exercise there is protection against fatty acid-induced insulin resistance and that this protective effect of exercise is accompanied by an increased lipogenic capacity of muscle and a resulting increase in partitioning of excess fatty acids toward triglyceride synthesis in muscle. This repartitioning of fatty acids after exercise toward intramyocellular triglyceride (IMTG) synthesis and oxidation reduced the accumulation of bioactive fatty acid metabolites, which are known to increase the activation of proinflammatory pathways in skeletal muscle and induce insulin resistance (Schenk & Horowitz, 2007). Similar results were obtained by transgenic overexpression of diacylglycerol acyltransferase (DGAT1) in mouse skeletal muscle, which mitigated the detrimental effect of fatty acids, and protected mice against high-fat diet-induced insulin resistance (Liu et al. 2007).

Our data show that, at 2 h after exercise in DIO rats, insulin sensitivity and insulin signalling were completely normalized; however, at 16 h after exercise, despite the normal insulin sensitivity there is only a partial restoration of insulin signalling. These data suggest that the complete normalization, by acute exercise, of the insulin action in obesity induced by diet may be caused by other factors and may involve other pathways. It is important to mention that the doses of insulin used were 3–4 times higher than the dose required for maximal insulin signalling, suggesting a possibility of spare insulin signalling under these conditions. This may involve proteins of the insulin signalling pathway not investigated, such as aPKC (protein kinase C), or other pathways such as cbl associated protein (CAP)/Casitas b lineage lymphoma (Cbl), which have a controversial role in insulin-induced glucose uptake in skeletal muscle. Another possibility may be associated with the haemodynamic changes induced by exercise. It is known that a single bout of exercise decreases sympathetic activity and increases muscle blood flow during the postexercise period. It is interesting that, during hyperinsulinaemia after a single bout of exercise, sympathetic activity is lower and muscle vasodilation is higher. These haemodynamic changes may also contribute to the reversion of the insulin resistance (Bisquolo et al. 2005). These data are in accordance with previous data demonstrating that exercise improves insulin-induced glucose uptake, at least in part, as a result of haemodynamic adaptations.

One limitation of the proposed theory of exercise-induced insulin sensitization in this study is that iNOS is normally not expressed in muscle (Kapur et al. 1997), and its expression is only induced in states of insulin resistance. However, exercise can also improve insulin action in healthy muscle. Therefore, this theory may not provide a universal explanation and, hence, is probably only one of several factors involved in the improvement of insulin sensitivity induced by exercise in DIO.

In summary, a single bout of exercise reverses insulin sensitivity in DIO rats by improving the insulin signalling pathway, in parallel with a decrease in iNOS expression and in the S-nitrosation of IR/IRS1/Akt. These results provide new insights into the mechanism by which exercise restores insulin sensitivity in DIO rats.

Acknowledgments

The authors thank Mr Luiz Janeri, Mr Jósimo Pinheiro and Márcio Alves da Cruz for their technical assistance. This work was supported by Grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Pesquisa (CNPq).

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