IRS-1 and Akt

Toxicology and Applied Pharmacology 241 (2009) 101–110

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Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p

Peroxynitrite mediates muscle insulin resistance in mice via nitration of IRβ/IRS-1 and Akt Jun Zhou, Kaixun Huang ⁎ Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, 430074, PR China

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Article history: Received 26 May 2009 Revised 31 July 2009 Accepted 4 August 2009 Available online 12 August 2009 Keywords: Insulin resistance Diabetes mellitus Peroxynitrite Tyrosine nitration Insulin receptor Insulin receptor substrate-1

a b s t r a c t Accumulating evidence suggests that peroxynitrite (ONOO-) is involved in the pathogenesis of insulin resistance. In the current study, we investigated whether insulin resistance in vivo could be mediated by nitration of proteins involved in the early steps of the insulin signal transduction pathway. Exogenous peroxynitrite donated by 3-morpholinosydnonimine hydrochloride (SIN-1) induced in vivo nitration of the insulin receptor β subunit (IRβ), insulin receptor substrate (IRS)-1, and protein kinase B/Akt (Akt) in skeletal muscle of mice and dramatically reduced whole-body insulin sensitivity and muscle insulin signaling. Moreover, in high-fat diet (HFD)-fed insulin-resistant mice, we observed enhanced nitration of IRβ and IRS-1 in skeletal muscle, in parallel with impaired whole-body insulin sensitivity and muscle insulin signaling. Reversal of nitration of these proteins by treatment with the peroxynitrite decomposition catalyst FeTPPS yielded an improvement in whole-body insulin sensitivity and muscle insulin signaling in HFD-fed mice. Taken together, these findings provide new mechanistic insights for the involvement of peroxynitrite in the development of insulin resistance and suggest that nitration of proteins involved in the early steps of insulin signal transduction is a novel molecular mechanism of HFD-induced muscle insulin resistance. © 2009 Elsevier Inc. All rights reserved.

Introduction Diabetes mellitus is one of the most costly chronic diseases with an estimated worldwide prevalence of 170 million in 2002, which is expected to double by 2030 according to the World Health Organization (Wild et al., 2004). It is generally considered that the pathogenesis of this disease is multifactorial. Type 2 diabetes, accounting for 90% of diabetes, is characterized by peripheral insulin resistance with an insulin secretory defect that varies in severity. Insulin resistance is central to the clustering of multiple metabolic abnormalities and clinical syndromes. Although significant progress has been made by a number of investigators, molecular mechanisms underlying insulin resistance are not fully understood. In the past few years, there has been accumulating evidence supporting the important role of NO and NOrelated reactive nitrogen species (RNS) such as peroxynitrite in the pathogenesis of diabetes and diabetic complications.

Abbreviations: BSA, bovine serum albumin; ECL, enhanced chemiluminescence; HFD, high-fat diet; iNOS, inducible nitric oxide synthase; IP, immunoprecipitation; IR, insulin receptor; IRS, insulin receptor substrate; NC, normal chow; NOS, nitric oxide synthase; NT, nitrotyrosine; ONOO-, peroxynitrite; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PI3-K, phosphatidylinositol 3-kinase; PTPase, phosphotyrosine phosphatase; PTP1B, protein tyrosine phosphatase 1B; pY, phosphotyrosine; RNS, reactive nitrogen species; SD, standard deviation; SIN-1, 3-morpholinosydnonimine hydrochloride; TTBS, Tris-buffered saline with Tween; WB, Western blotting. ⁎ Corresponding author. Fax: +86 27 87543632. E-mail address: [email protected] (K. Huang). 0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2009.08.005

Peroxynitrite (ONOO-) is the product of the near diffusionlimited reaction between superoxide and nitric oxide (Beckman, 1996; Koppenol, 1998) and is believed to be at least partially responsible for the toxic and detrimental effects of nitric oxide in biological systems (Beckman and Koppenol, 1996). It has been estimated that steady-state concentrations of peroxynitrite may be significant in the vicinity of activated macrophages that generate superoxide and nitric oxide concomitantly (Ischiropoulos et al., 1992). While generation of peroxynitrite may be beneficial in terms of host defense against invading microorganisms, excess peroxynitrite may be detrimental and entails damage to biomolecules, because peroxynitrite is a powerful oxidizing and nitrating species, causing DNA damage, lipid peroxidation, oxidation of protein-associated thiol groups, and nitration of protein tyrosine residues (Beckmann et al., 1994; Denicola and Radi, 2005; Radi et al., 1991a, 1991b). Tyrosine nitration is commonly and widely studied and has been shown in a number of instances to affect protein function, e.g., it can deactivate enzymes such as succinylCoA:3-oxoacid CoA-transferase (Turko et al., 2001) and tyrosine hydroxylase (Blanchard-Fillion et al., 2001). Tyrosine nitration has been associated with a large variety of inflammatory, cardiovascular and neurodegenerative diseases including, for example, retinal ischemia, lung infection, cardiovascular inflammation, atherosclerosis, diabetes, Parksinson's disease, Alzheimer's disease, and Huntington's disease (Halliwell et al., 1999; Turko and Murad, 2002).

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Recently, increased nitrotyrosine formation has been documented in both experimental diabetic animals (Garcia Soriano et al., 2001; Ishii et al., 2001; Pacher et al., 2005; Reddy and Bradley, 2004; Suarez-Pinzon et al., 1997; Szabo et al., 2002a, 2002b) and diabetic patients (Ceriello et al., 2001; Szabo et al., 2002a, 2002b; Thuraisingham et al., 2000; Torres et al., 2004). Moreover, nitrotyrosine formation is increased in insulin-resistant animals and humans. Increased nitrotyrosine formation has been observed not only in high-fat diet (HFD)-fed insulin-resistant rodents (Molnar et al., 2005; Yamaguchi et al., 2006) but also in the plasma (Ceriello et al., 2001) and skeletal muscle tissue of patients suffering from type 2 diabetes (Torres et al., 2004). Protein tyrosine nitration alters the structures and functions of proteins and therefore may prevent tyrosine phosphorylation (Greenacre and Ischiropoulos, 2001; Ischiropoulos, 1998). As a consequence, peroxynitrite may affect insulin signal transduction through nitration of key tyrosine residues on the proteins involved in insulin signaling. In line with this concept, Nomiyama et al. (2004) have shown that peroxynitrite impairs insulin action in vitro by decreasing tyrosine phosphorylation of insulin receptor substrate (IRS)-1 concurrent with nitration of its tyrosine residues. Moreover, the involvement of peroxynitrite in the development of insulin resistance in mice has been recently demonstrated. Duplain et al. (2008) have provided in vivo evidence that high concentrations of peroxynitrite are formed and are responsible for HFD-induced insulin resistance in skeletal muscle by increasing Akt nitration. However, the in vivo effect of peroxynitrite on insulin signal merits further investigation. At present, it remains unknown whether the insulin receptor (IR), IRS-1 or phosphatidylinositol 3-kinase (PI3-K) are targets of protein nitration in vivo and whether insulin resistance in vivo could be mediated by nitration of proteins involved in the early steps of the insulin signal transduction pathway. Skeletal muscle accounts for the majority of insulin-regulated whole-body glucose disposal. Up to 75% of insulin-dependent glucose disposal occurs in skeletal muscle, whereas adipose tissue accounts for only a small fraction (Klip and Paquet, 1990). In the present study, we investigated the effect of SIN-1, a constitutive producer of peroxynitrite, on insulin signal in skeletal muscle of mice. Furthermore, we investigated whether HFD-induced insulin resistance in mice could be mediated by nitration of proteins involved in the early steps of the insulin signal transduction pathway by using FeTPPS, a peroxynitrite decomposition catalyst (Misko et al., 1998). Our findings provide new mechanistic insights for the involvement of peroxynitrite in the development of insulin resistance and suggest that nitration of proteins involved in the early steps of the insulin signal transduction pathway is a novel molecular mechanism of HFDinduced muscle insulin resistance.

(Japan). All other chemicals were of the highest commercial grade available. Animals. Five-week-old male C57BL/6 mice were housed in a temperature (22 ± 3 °C)- and humidity (50% ± 20%)-controlled room with a 12-h day/night cycle. The mice were fed with either high-fat diet (HFD; energy content: 72% fat, 28% protein and b1% carbohydrate) or normal chow (NC) for 8 weeks (Cook et al., 2004). The nitrate content of both diets was comparable (50–60 mg/kg). Throughout the study period, mice were allowed free access to food and water ad libitum. Food intake was recorded daily, and body weight was recorded three times per week. During the last 5 days of the diet period, mice received an intraperitoneal injection of 10 mg/ kg FeTPPS (dissolved in normal saline), a peroxynitrite decomposition catalyst, or saline (Duplain et al., 2008). Animal experimentation was in accordance with institutional guidelines. SIN-1 treatment. Acute SIN-1 treatment was performed in normal mice 30 min before muscle extraction. Briefly, mice were fasted for 6 h and then injected intraperitoneally with either various doses of SIN-1 (5, 10 mg/kg body weight) or vehicle (50 mM PBS, pH 7.4). Chronic treatment was performed by 10 mg/kg SIN-1 intraperitoneally injected every 2 h, until completing four doses in 8 h. Blood chemical analysis. Glucose and insulin plasma concentrations were measured after a 6-h fast in blood samples obtained by retroorbital puncture. Whole-blood glucose was measured with a blood glucose meter and blood glucose test strips (Roche Diagnostic Corp., Indianapolis, IN, USA). Plasma insulin was determined by using an insulin ELISA kit with mouse insulin as a standard (Crystal Chem, Downers Grove, IL). The insulin tolerance test. On the second day following the fasting glucose and insulin measurements, mice were fasted for 6 h and submitted to an insulin tolerance test as described previously (Carvalho-Filho et al., 2005). Briefly, mice were injected intraperitoneally with 1.5 IU/kg insulin, blood was collected from the tip of the tail at 0 (basal), 5, 10, 15, 20, 25, and 30 min thereafter, and glucose was measured with a blood glucose meter and blood glucose test strips. Glucose disappearance rate (Kitt) was calculated from the formula 0.693/t1/2. The glucose t1/2 was calculated from the slope of the least square analysis of blood glucose concentration during the linear phase of decline (Bonora et al., 1989). Protein extraction, immunoprecipitation, and Western blotting analysis. Mice were intraperitoneally injected with either saline or insulin (5 U/kg) (Duplain et al., 2008), and 5 min later, mice were

Materials and methods Reagents and antibodies. Bovine serum albumin (BSA), 3-morpholinosydnonimine hydrochloride (SIN-1), and anti-phosphotyrosine antibody were purchased from Sigma Co. Protease inhibitor cocktail and FeTPPS were obtained from Calbiochem. Antinitrotyrosine antibody and anti-p85 antibody were obtained from Upstate Biotechnology Incorporated (Lake Placid, NY). Anti-IR β subunit (IRβ) antibody, anti-IRS-1 antibody, anti-Akt antibody, and protein A/G plus-Agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Akt (ser473) antibody was from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were from Pierce Company. Porcine insulin (28 IU/mg) was obtained from Wanbang Pharm (Xuzhou, China). TRIzol reagent was obtained from Invitrogen. Molony murine leukemia virus (M-MLV) reverse transcriptase (200U) and oligo (dT) were purchased from Promega. 10 mM dNTP was from Roche. 2 ×SYBR Green PCR Master Mix was obtained from Toyobo

Fig. 1. Effect of SIN-1 on whole-body insulin sensitivity in mice. Mice were injected intraperitoneally with either indicated doses of SIN-1 or vehicle, and thereafter glucose disappearance rates were measured by the 30-min insulin tolerance test. Values are expressed as means ± SD from six mice. ⁎⁎P b 0.01 versus vehicle control group.

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sacrificed by cervical dislocation under diethyl ether anesthesia. The soleus muscles were immediately isolated, rinsed in phosphatebuffered saline (PBS, pH 7.4), flash frozen in liquid nitrogen, and

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stored at −80 °C until analysis. The muscles were homogenized in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5 % sodium deoxycholate, 10 mM NaF, 1 mM Na3VO4, pH 7.5) containing

Fig. 2. Effect of SIN-1 on nitration of IRβ, IRS-1, and Akt and nitrite/nitrate levels in muscle of mice. Mice were subjected to acute or chronic SIN-1 treatment (10 mg/kg), and thereafter nitration of IRβ, IRS-1, and Akt in muscle was measured by Western blot analysis with anti-nitrotyrosine antibody following immunoprecipitation with indicated antibodies. (A) Nitration of IRβ after acute 30-min SIN-1 treatment. (B) Nitration of IRS-1 after acute and chronic SIN-1 treatment. (C) IRS-1 protein level after acute and chronic SIN1 treatment. (D) Nitration of Akt after acute 30-min SIN-1 treatment. (E) Muscle nitrite/nitrate levels after acute 30-min SIN-1 treatment. Values are expressed as means ± SD from six mice. ⁎P b 0.05, ⁎⁎P b 0.01 versus vehicle control group. IP, immunoprecipitation; WB, Western blotting; nY, nitrotyrosine.

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10 μl/ml protease inhibitor cocktail, centrifuged at 13,000 × g for 15 min, and the protein content of the soluble fraction was determined by the Bradford method using BSA as standard. For immunoprecipitation experiments, muscle lysates containing 1 mg of total protein were incubated with 4 μg of anti-IRβ, anti-IRS-1 or anti-Akt antibodies for 1 h at 4 °C. After collection on protein A/G plus-Agarose, the immune complexes were washed three times with lysis buffer, boiled in Laemmli buffer for 5 min, and thereafter subjected to Western blotting analysis. Protein samples from whole-tissue extracts or immunoprecipitates were separated on SDS-polyacrylamide gels, and then transferred to nitrocellulose membranes for Western analysis. Membranes were blocked with 5% nonfat milk T-TBS solution, and incubated with the indicated antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase. Antibody labeling was visualized with ECL detection kit (Pierce) according to the manufacturer's instructions, and the images were captured on a KODAK X-ray film using a KODAK automatic film processor (Kodak, Rochester, NY, USA). PI3-kinase assay. PI3-kinase activity in immunoprecipitates with anti-IRS-1 was measured, as previously described with minor modifications (Yasukawa et al., 2005). Briefly, in vitro phosphorylation reaction of phosphatidylinositol (Sigma) was performed in PI3kinase buffer (100 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 15 mM MgCl2, 70 μM ATP) containing 10 μCi of [γ-32P]ATP at 37 °C for 10 min. The reaction was stopped by the addition of 8 N HCl. The phospholipids were extracted with chloroform/methanol (1:1, v/v). The organic phase, containing the labeled PI3-kinase products, was separated by silica gel thin-layer chromatography plates. The plates were then developed in CHCl3/CH3OH/H2O/NH4OH (60:47:11:3.2), dried, and visualized by autoradiography. Real-time RT-PCR analysis. Total RNA was isolated from the soleus muscles using TRIzol reagent according to the manufacturer's instructions. Complementary DNA (cDNA) was prepared from the RNA in the presence of M-MLV reverse transcriptase, dNTP, and oligo (dT) in Tris(hydroxymethyl) aminomethane hydrochloride (Tris– HCl) buffer (50 mM, pH 8.3). The reaction mixture was incubated at 37 °C for 60 min and stopped by heating at 95 °C for 10 min. Real-time quantitative PCR was performed with a DNA Engine Opticon 2 (MJ Research, Watertown, Massachusetts, USA), using the SYBR Green PCR Master Mix kit, according to the vendor’s protocol. The primers used for PCR were GAPDH 5′-ttcaccaccatggagaaggc-3′ (forward) and 5′-ggcatggactgtggtcatga-3′ (reverse), iNOS 5′cagctgggctgtacaaacctt-3′ (forward) and 5′-cattggaagtgaagcgtttcg-3′ (reverse) (Overbergh et al., 1999). All PCRs were performed in triplicate, and the thermal cycle conditions were as follows: 95 °C for 1 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Expression levels of iNOS were corrected by normalization to the expression levels of the housekeeping gene GAPDH, and relative expression levels were calculated with the 2-ΔΔCt rule (Livak and Schmittgen, 2001). Determination of nitrite and nitrate. NO levels were determined indirectly by quantification of their oxidized products of degradation (NOˉ2 and NOˉ3 ), using nitrate reductase and Griess reagent (Green et al., 1982) as previously described (Moshage et al., 1995). Briefly, 200 μl samples were incubated at 37 °C for 30 min in 100 mM Tris

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buffer (pH 7.4) containing 1 mM FAD, 10 mM NADPH, and 10 U/ml nitrate reductase. LDH (1000 U/ml) and 0.02 M sodium pyruvate were added to each mixture, and the incubation was continued for 15 min. The Griess reagent was added, and after 10 min, the nitrite content was determined colorimetrically at 546 nm. The standard curve of sodium nitrite was plotted to calculate concentration of nitrite/nitrate. Statistical analysis. Data are expressed as means ± SD. Statistical analysis was performed by the two-tailed Student's t-test or by ANOVA (one-way or two-way ANOVA) followed by Tukey post hoc test, if appropriate. A value of P b 0.05 was considered statistically significant. Results SIN-1 induces insulin resistance in muscle of mice by means of nitration Male C57BL/6 mice were treated with various doses of SIN-1 to investigate whether this peroxynitrite donor could induce insulin resistance in vivo. Thirty minutes after SIN-1 administration (10 mg/ kg body weight), an insulin resistance condition was established, as indicated by a markedly lower plasma glucose disappearance rate after the 30-min insulin tolerance test (Kitt) (P b 0.01, Fig. 1). Nevertheless, SIN-1 at a dose of 5 mg/kg body weight failed to significantly lower plasma glucose disappearance rate. We hypothesized that peroxynitrite can modulate insulin action in muscle by inducing nitration of proteins involved in the early steps of insulin signaling. The ability of SIN-1 to induce nitration of IRβ in muscle was investigated by immunoprecipitation with anti-IRβ antibody and immunoblotting with anti-nitrotyrosine antibody. The results showed that SIN-1-induced insulin resistance was associated with enhanced nitration of IRβ (Fig. 2A). Moreover, treatment with SIN-1 for 30 min also led to nitration of IRS-1, as demonstrated by immunoprecipitation with anti-IRS-1 antibody and immunoblotting with anti-nitrotyrosine antibody (Fig. 2B), but did not change IRS-1 protein levels (Fig. 2C) and IRS-1 serine phosphorylation levels (data not shown). However, when SIN-1 was given during 8 h, the increased nitration of IRS-1 (Fig. 2B) was associated with a reduced concentration of this protein in the muscle (Fig. 2C). Furthermore, we also observed enhanced nitration of Akt after acute treatment with SIN-1 (Fig. 2D). Nevertheless, we did not observe nitration of p85 subunit of PI3-K (data not shown). In addition, we measured the nitrite and nitrate levels in skeletal muscle. As shown in Fig. 2E, the muscle nitrite and nitrate levels were markedly increased by 91% in SIN-1-treated mice as compared with controls (P b 0.01). The effect of SIN-1 on IRβ and IRS-1 tyrosine phosphorylation and Akt serine phosphorylation was investigated in soleus muscle. We demonstrated that SIN-1 administration to mice induced a ∼40% reduction in insulin-induced IRβ and IRS-1 tyrosine phosphorylation, and p85 association with IRS-1 (Figs. 3A–C). Furthermore, SIN-1 administration to mice induced a ∼ 46% reduction in insulin-induced PI3-K activity (Fig. 3E). Likewise, SIN-1 treatment also reduced insulin-induced Akt serine phosphorylation by 52% (Fig. 3D). Diet-induced insulin resistance is associated with enhanced nitration Physiological parameters of the mice fed with NC or HFD are shown in Table 1. After 8 weeks of diet, the body weight of HFD-fed

Fig. 3. Effect of SIN-1 on phosphorylation of IRβ, IRS-1, and Akt and PI3-kinase activity in muscle of mice. Mice were subjected to acute 30-min SIN-1 treatment (10 mg/kg) followed by insulin stimulation (5 U/kg), and thereafter phosphorylation of IRβ, IRS-1, and Akt in muscle was measured by Western blot analysis following immunoprecipitation with indicated antibodies. (A) Tyrosine phosphorylation of IRβ after acute 30-min SIN-1 treatment. (B) Tyrosine phosphorylation of IRS-1 after acute 30-min SIN-1 treatment. (C) p85 association with IRS-1 after acute 30-min SIN-1 treatment. (D) Serine phosphorylation of Akt after acute 30-min SIN-1 treatment. (E) IRS-1 associated PI3-kinase activity after acute 30-min SIN-1 treatment. Values are expressed as means ± SD from six mice. ⁎P b 0.05, ⁎⁎P b 0.01 versus vehicle control group. IP, immunoprecipitation; WB, Western blotting; pY, phosphotyrosine.

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Table 1 Physiological parameters of mice fed with NC or HFD. Parameter

NC

HFD

HFD+FeTPPS

Body weight (g) Blood glucose (mM) (fasted) Plasma insulin (pM) (fasted) Food intake (g/day) Liver weight (g) Epididymal adipose issue (g)

26.9 ± 0.7 7.0 ± 0.5 243 ± 36 3.22 ± 0.07 1.31 ± 0.08 0.39 ± 0.04

29.6 ± 0.6⁎⁎ 10.2 ± 0.9⁎⁎ 369 ± 43⁎⁎ 3.56 ± 0.08⁎⁎ 1.54 ± 0.09⁎⁎ 0.82 ± 0.07⁎⁎

29.4 ± 0.7⁎⁎ 7.9 ± 0.7## 247 ± 31## 3.54 ± 0.09⁎⁎ 1.53 ± 0.08⁎⁎ 0.79 ± 0.08⁎⁎

Data are expressed as means ± SD from six mice. ⁎P b 0.05, ⁎⁎P b 0.01 versus NC-fed mice; #P b 0.05, ##P b 0.01 versus HFD-fed mice.

mice was significantly greater as compared with NC-fed mice (P b 0.01), and this was associated with increased liver and epididymal adipose accretion in the former group (P b 0.01). HFD markedly increased the fasting glucose (P b 0.01) and insulin plasma concentrations (P b 0.01). Moreover, HFD-induced insulin resistance was demonstrated by a reduction in the Kitt of obese animals (P b 0.01, Fig. 4A). The effect of HFD on iNOS mRNA expression and nitrite and nitrate levels in skeletal muscle of mice was also investigated. iNOS mRNA expression was increased by 1.4-fold in skeletal muscle of HFDfed mice as compared with NC-fed mice (Fig. 4B), similar to previous observations (Perreault and Marette, 2001). Furthermore, the nitrite and nitrate levels were increased by 81% in skeletal muscle of HFD-fed mice as compared with NC-fed mice (Fig. 4C). To investigate whether HFD-induced insulin resistance could be mediated by nitration of proteins involved in the early steps of insulin signaling, soleus muscles of HFD-fed mice were removed, and nitrated proteins were determined and compared with those of controls that received normal chow for the same period. We observed a marked increase in nitration of IRβ and IRS-1 in the muscle of obese animals (P b 0.01, Figs. 5A and B). Insulin-stimulated tyrosine phosphorylation of IRβ was reduced in the skeletal muscle of obese animals by 36% (P b 0.01, Fig. 5A). Moreover, insulin-stimulated IRS-1 tyrosine phosphorylation was also reduced by 39% when compared with controls (P b 0.01, Fig. 5B). In consequence, insulin-stimulated p85 association with IRS-1 and PI3-K activity were significantly reduced by 43% and 45% in skeletal muscle of HFD-fed mice as compared with controls, respectively (P b 0.01, Fig. 5C).

FeTPPS-treated obese mice (Figs. 5A and B). Likewise, FeTPPS treatment also restored insulin-stimulated p85 association with IRS1 and IRS-1 associated PI3-K activity in skeletal muscle (Fig. 5C). Discussion Nitric oxide synthases (NOSs) are the exclusive source of NO in mammals. Among the three isoforms of NOS, inducible NOS (iNOS) is known to produce far larger amounts of NO in pathological states compared with the other isoforms, and iNOS has been recognized to play a role in the pathogenesis of inflammatory and autoimmune diseases (Nathan, 1997; Stichtenoth and Frolich, 1998). There is growing evidence suggesting that iNOS plays a major role in the

FeTPPS reduces nitration of IRβ/IRS-1 and improves insulin sensitivity in diet-induced obesity Male C57BL/6 mice received a high-fat diet for 8 weeks, and a subgroup of these animals received FeTPPS at 10 mg/kg body weight for 5 days. FeTPPS markedly decreased the blood glucose and plasma insulin concentrations as compared with HFD-fed mice (P b 0.01, Table 1), but FeTPPS had no effect on body weight, food intake, liver, and epididymal adipose weight of HFD-fed mice. Consistent with previous findings (Duplain et al., 2008), the glucose disappearance rate at 30 min in the insulin tolerance test was significantly lower in mice with diet-induced obesity, and this reduction was reversed by FeTPPS treatment (Fig. 4A), indicating an improvement in insulin sensitivity. In addition, the effect of FeTPPS on iNOS mRNA expression and nitrite and nitrate levels in skeletal muscle of mice was also investigated. FeTPPS did not decrease iNOS mRNA expression in skeletal muscle of HFD-fed mice (Fig. 4B). However, the nitrite and nitrate levels in skeletal muscle were notably increased in FeTPPS-treated obese mice as compared with HFD-fed mice (P b 0.01, Fig. 4C). Furthermore, the effect of FeTPPS treatment on the nitration and phosphorylation of proteins involved in the early steps of insulin signaling was investigated. The results demonstrated that FeTPPS treatment attenuated the nitration of IRβ and IRS-1 in the muscle of mice (Figs. 5A and B). Moreover, the reduced insulin-stimulated IRβ and IRS-1 tyrosine phosphorylation was reversed in the muscle of

Fig. 4. Effect of FeTPPS on whole-body insulin sensitivity, muscle iNOS mRNA expression, muscle nitrite and nitrate levels in HFD-fed obese mice. (A) Whole-body insulin sensitivity of NC-fed mice, HFD-fed mice and HFD-fed mice treated with FeTPPS (10 mg/kg), as evaluated by glucose disappearance rates and measured by the 30-min insulin tolerance test. (B) iNOS mRNA expression in skeletal muscle of NC-fed mice, HFD-fed mice and HFD-fed mice treated with FeTPPS (10 mg/kg). (C) nitrite and nitrate levels in skeletal muscle of NC-fed mice, HFD-fed mice and HFD-fed mice treated with FeTPPS (10 mg/kg). Values are expressed as means ± SD from six mice. ⁎P b 0.05, ⁎⁎P b 0.01 versus NC; #P b 0.05, ##P b 0.01 versus HFD.

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Fig. 5. Effect of FeTPPS on nitration of IRβ/IRS-1, and muscle insulin signaling in HFD-fed obese mice. (A) Nitration and insulin-stimulated tyrosine phosphorylation of IRβ in muscle of mice, as measured by immunoprecipitation with anti-IRβ antibody and immunoblotting with anti-nitrotyrosine and anti-phosphotyrosine antibodies. (B) Nitration and insulin-stimulated tyrosine phosphorylation of IRS-1 in muscle of mice, as measured by immunoprecipitation with anti-IRS-1 antibody and immunoblotting with antinitrotyrosine and anti-phosphotyrosine antibodies. (C) Insulin-stimulated p85 association with IRS-1 and PI3-kinase activity in muscle of mice, as measured by immunoprecipitation with anti-IRS-1 antibody and immunoblotting with anti-p85 antibody, as well as a PI3-kinase activity assay. Values are expressed as means ± SD from six mice. ⁎P b 0.05, ⁎⁎P b 0.01 versus NC; #P b 0.05, ##P b 0.01 versus HFD. IP, immunoprecipitation; WB, Western blotting; nY, nitrotyrosine; pY, phosphotyrosine.

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development of insulin resistance associated with obesity and other inflammatory settings (Marette, 2002; Perreault and Marette, 2001). However, it remains unclear how iNOS activity and nitric oxide production cause insulin resistance in muscle. Recently, the effect of iNOS has been demonstrated to be associated with direct modification of insulin signaling components by RNS. In this regard, Nomiyama et al. (2004) showed that peroxynitrite, an end-product of iNOS, impairs insulin action in vitro by decreasing tyrosine phosphorylation of IRS-1 concurrent with nitration of its tyrosine residues. Moreover, the Snitrosylation of IR, IRS-1, and Akt has been shown to be responsible for impaired insulin signaling in cells and isolated muscle exposed to NO donors, as well as in muscle of genetic and nutritional insulinresistant mouse models (Carvalho-Filho et al., 2005; Kaneki et al., 2007; Yasukawa et al., 2005). A recent in vivo study showed that HFD reduced insulin-stimulated Akt phosphorylation and induced insulin resistance in skeletal muscle by increasing nitrotyrosine content and Akt nitration, which was abolished by treatment with peroxynitrite decomposition catalyst FeTPPS (Duplain et al., 2008). In the present study, we demonstrated that proteins involved in early steps of insulin signal transduction can be nitrated and that the nitration of the IR and IRS-1 results in reduced tyrosine phosphorylation, association of p85 with IRS-1 and IRS-1 associated PI3-K activity, suggesting a novel mechanism for iNOS-induced insulin resistance in muscle. Initially, we demonstrated that SIN-1, a peroxynitrite donor, dramatically reduced whole-body insulin sensitivity in normal mice assessed by insulin tolerance tests. The effect of SIN-1 was associated with increased nitration of IRβ in skeletal muscle. This posttranslational modification was accompanied by a marked reduction in insulin-induced IRβ autophosphorylation. Moreover, the administration of SIN-1 also led to nitration of IRS-1 in skeletal muscle, which was accompanied by a reduction of insulin-induced IRS-1 phosphorylation, p85 association with IRS-1 and IRS-1 associated PI3-kinase activity. Interestingly, the administration of this peroxynitrite donor for 8 h, which allows a more prolonged nitration of IRS-1, is also accompanied by a reduction in IRS-1 protein content. An earlier report demonstrated that the administration of SIN-1 to 3T3-L1 adipocytes induced nitration and degradation of IRS-1, and consequently reduced protein content (Nomiyama et al., 2004). Taken together, these data suggest that an increase in IRS-1 nitration may favor its degradation and explain the reduced protein content observed in the muscle of mice that received the peroxynitrite donor. This may be important, since in some situations of insulin resistance, there is a reduction in IRS-1 expression in muscle, contributing to reduced insulin sensitivity (Saad et al., 1993; Zecchin et al., 2003). In addition, SIN-1 administration also led to Akt nitration, which was accompanied by a reduction of insulin-stimulated Akt serine phosphorylation. The nitration of IRβ, IRS-1and Akt is associated with an important reduction of insulin signaling in muscle, which may play an important role in the reduced whole-body insulin sensitivity in mice that received this drug. However, further studies are necessary to elucidate this point. For instance, the determination of the muscle glucose uptake ex vivo following SIN-1 administration will help to prove the muscle implication in the model. It is generally accepted that several molecular mechanisms related to insulin resistance are increased phosphotyrosine phosphatase (PTPase) activity, mainly protein tyrosine phosphatase 1B (PTP1B) (Elchebly et al., 1999), increased serine phosphorylation of IRSs (Hotamisligil et al., 1996), and increased S-nitrosylation of IRβ, IRS-1, and Akt (Carvalho-Filho et al., 2005; Yasukawa et al., 2005). The present study suggests that nitration of proteins involved in early steps of insulin signal is a novel mechanism of insulin resistance in vivo. The insulin resistance induced by nitration seems to occur in at least three different steps of insulin action, i.e., by reducing IRβ autophosphorylation, by reducing IRS-1 protein content and tyrosine phosphorylation, and by reducing Akt serine phosphorylation. It is worth noting that SIN-1 may produce nitric oxide as well as peroxynitrite. The present study

also demonstrates that SIN-1 induces an increase in NO levels in skeletal muscle as evidenced by elevated nitrite and nitrate levels. It is likely that NO released by SIN-1 causes the S-nitrosylation of IR, IRS-1 and Akt, and a defect in muscle insulin signaling as reported earlier (Carvalho-Filho et al., 2005), thus contributing to muscle insulin resistance. Further studies are necessary to demonstrate the involvement of S-nitrosylation in this model. High blood levels of free fatty acids and glucose, which are often found in diabetes and obesity, can induce the production of superoxide through various mechanisms including increased uptake of substrates by the mitochondria (Nishikawa et al., 2000). Consequently, when iNOS expression and NO levels are increased in muscle of HFD-induced obese mice, peroxynitrite may be easily produced in skeletal muscle by the reaction of NO with superoxide. In line with this, Duplain et al. (2008) demonstrated that peroxynitrite is produced in skeletal muscle of HFD-fed obese mice and contributes to muscle insulin resistance by increasing Akt nitration and reducing Akt phosphorylation. Here we investigated whether nitration of IRβ and IRS-1 could also be observed in the muscle of animal model of insulin resistance. In HFD-fed mice, we observed a remarkable reduction in whole-body insulin sensitivity, with a parallel increase in body weight, blood glucose, and plasma insulin concentrations, consistent with previous studies (Duplain et al., 2008; Perreault and Marette, 2001). Moreover, there was a parallel increase in the nitration of IRβ and IRS-1 in skeletal muscle. As previously described (Duplain et al., 2008; Perreault and Marette, 2001), we also observed impaired insulin signaling in the muscle of HFD-fed mice. In HFD-fed mice, the reduction of peroxynitrite content by the peroxynitrite decomposition catalyst FeTPPS is accompanied by a reduction of nitration and improvement of muscle insulin signaling through IRβ and IRS-1, as well as a reversal of whole-body insulin resistance. Taken together, these data demonstrate that the insulin resistance observed in HFD-fed mice is accompanied by an increase in nitration of proteins involved in early steps of insulin action. Moreover, in HFDfed mice, the reduction in peroxynitrite content is followed by the reduction in nitration of these proteins and reversal of insulin resistance. In line with this, Duplain et al. (2008) demonstrated that FeTPPS reduced peroxynitrite content and Akt nitration, and restored insulin-stimulated Akt phosphorylation and insulin-stimulated glucose uptake in isolated skeletal muscle from HFD-fed mice. It is noteworthy that FeTPPS does not alter muscle iNOS mRNA expression, body weight or food intake of HFD-fed mice, but dramatically increases nitrite and nitrate levels in skeletal muscle of HFD-fed mice, probably due to its peroxynitrite decomposition reactivities. It has been demonstrated that FeTPPS catalytically decomposes peroxynitrite under physiologically relevant conditions and results in a net isomerization of peroxynitrite to nitrate (Jensen and Riley, 2002). Accordingly, it seems reasonable to presume that the elevated nitrite and nitrate levels in FeTPPS-treated obese mice result from peroxynitrite decomposition catalyzed by FeTPPS. Taken together, we and others have shown that decomposition of peroxynitrite by FeTPPS prevents whole-body and skeletal muscle insulin resistance in mice rendered obese by feeding a high-fat diet, demonstrating that tyrosine nitration makes a significant contribution to iNOS-mediated insulin resistance in this model. In addition, IRS-1 protein expression was not altered by HFD in the present study, which is consistent with the findings of Perreault and Marette (2001). Nonetheless, we cannot exclude the possibility that a longer duration of HFD may reduce IRS-1 protein expression, as demonstrated by Carvalho-Filho et al. (2005). In addition, a 30-min insulin tolerance test is applied to quickly assess insulin sensitivity in the present study, but further studies are necessary to measure the changes in peripheral glucose disposal with FeTPPS over time. A longer insulin tolerance test will help to assess the full scale of FeTPPS effects over time. Other molecular mechanisms of insulin resistance have been demonstrated in the muscle of genetic and nutritional insulin-

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resistant animal models, including an increase in IRS serine phosphorylation, an increase in protein tyrosine phosphatase 1B activity and also an increase in the S-nitrosylation of IRβ, IRS-1, and Akt (Carvalho-Filho et al., 2005; Elchebly et al., 1999; Hotamisligil et al., 1996). It is possible that multiple mechanisms can contribute to insulin resistance, and reversal of one of them can improve insulin action, as we demonstrate here and as previously demonstrated for other mechanisms (Carvalho-Filho et al., 2005, 2006; Duplain et al., 2008; Elchebly et al., 1999; Hirosumi et al., 2002; Klaman et al., 2000; Yasukawa et al., 2005). It is worth noting that peroxynitrite can potentially impact directly upon at least some of these mechanisms. For instance, peroxynitrite can inhibit PTP1B activity in vitro as reported earlier (Takakura et al., 1999), although the effect of peroxynitrite on PTP1B activity in vivo remains to be determined. However, peroxynitrite does not appear to alter serine phosphorylation (Ser307) of IRS-1 (Nomiyama et al., 2004). In summary, the results presented herein may represent a potential mechanism involved in iNOS-induced insulin resistance. The increase in iNOS expression provokes enhanced production of peroxynitrite that can induce nitration of IRβ, IRS-1, and Akt, resulting in downregulation of insulin signaling. Moreover, our data raise the possibility that agents that reduce peroxynitrite formation and, consequently, reduce nitration might have beneficial effects on obesity-linked insulin resistance and associated complications. Conflict of interest statement The authors declare that there are no conflicts of interest.

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