Antioxidants preserve redox balance and inhibit c-Jun-N-terminal kinase pathway while improving insulin signaling in fat-fed rats: evidence for the role of oxidative stress on IRS-1 serine phosphorylation and insulin resistance

in Journal of Endocrinology

The oxidative stress-sensitive c-Jun-N-terminal kinase (JNK) pathway is known to be activated in diabetic condition and is involved in the progression of insulin resistance. However, the effect of antioxidants on JNK pathway and insulin resistance has not been investigated. The present study was aimed to investigate the effect of antioxidants on redox balance, insulin sensitivity, and JNK pathway in high-fat-fed rats. Male Wistar rats were divided into four groups: the control group – received a rodent chow; control+antioxidant group – fed with rodent chow supplemented with 0.2% (w/w) vitamin E, 0.3% (w/w) vitamin C, and 0.5% (w/w) α-lipoic acid; high-fat group – received high-fat diet; and high fat+antioxidant group – fed with high-fat diet supplemented with above antioxidants. Fat feeding to rats for 9 weeks significantly increased IRS-1 serine phoshorylation, reduced insulin-stimulated IRS-1 tyrosine phosphorylation and insulin sensitivity. High-fat diet also impaired redox balance and activated the redox-sensitive serine kinase – JNK pathway. Antioxidant supplementation along with high-fat diet preserved the free radical defense system, inhibited the activation of JNK pathway, and improved insulin signaling and insulin sensitivity. The present study shows for the first time that antioxidants inhibit JNK pathway and IRS-1 serine phosphorylation while improving insulin sensitivity in fat-fed rats. These findings implicate the beneficial effect of antioxidants in obesity-/dyslipidemia-induced insulin resistance in humans.

Abstract

The oxidative stress-sensitive c-Jun-N-terminal kinase (JNK) pathway is known to be activated in diabetic condition and is involved in the progression of insulin resistance. However, the effect of antioxidants on JNK pathway and insulin resistance has not been investigated. The present study was aimed to investigate the effect of antioxidants on redox balance, insulin sensitivity, and JNK pathway in high-fat-fed rats. Male Wistar rats were divided into four groups: the control group – received a rodent chow; control+antioxidant group – fed with rodent chow supplemented with 0.2% (w/w) vitamin E, 0.3% (w/w) vitamin C, and 0.5% (w/w) α-lipoic acid; high-fat group – received high-fat diet; and high fat+antioxidant group – fed with high-fat diet supplemented with above antioxidants. Fat feeding to rats for 9 weeks significantly increased IRS-1 serine phoshorylation, reduced insulin-stimulated IRS-1 tyrosine phosphorylation and insulin sensitivity. High-fat diet also impaired redox balance and activated the redox-sensitive serine kinase – JNK pathway. Antioxidant supplementation along with high-fat diet preserved the free radical defense system, inhibited the activation of JNK pathway, and improved insulin signaling and insulin sensitivity. The present study shows for the first time that antioxidants inhibit JNK pathway and IRS-1 serine phosphorylation while improving insulin sensitivity in fat-fed rats. These findings implicate the beneficial effect of antioxidants in obesity-/dyslipidemia-induced insulin resistance in humans.

Introduction

The incidence of type 2 diabetes is rapidly increasing in all parts of the world. It has been estimated that over 200 million people will be afflicted with the disease by the end of this decade. The pathogenesis of type 2 diabetes is not understood in any great detail, but it has become increasingly clear that acquired and non-genetic factors represent a critical link in the pathogenesis of type 2 diabetes (Saltiel & Kahn 2001, Leng et al. 2004). The previous reports have shown obesity as a primary contributor to the acquired insulin resistance (Spiegelman & Flier 2001). Numerous studies have documented the development of insulin resistance as a result of increased intake of dietary fats. In both humans and animals, there is an inverse relationship between fasting plasma triglyceride concentration and insulin sensitivity (Perseghin et al. 1997). There is an even stronger relationship between the accumulation of intracellular triglycerides and insulin resistance (Phillips et al. 1996, Pan et al. 1997, Krssak et al. 1999). Insulin resistance and several defects in insulin signaling have been reported in rats fed with high-fat diet. The high-fat diet-fed rodent is a model of research interest because it might provide insights into the mechanisms underlying insulin resistance in obese individuals with impaired glucose tolerance. Even though insulin resistance in obesity has been demonstrated, the molecular basis for the obesity and/or high-fat-induced insulin resistance is not clear.

Recently, we (Vinayagamoorthi et al. 2006) along with others (Rudich et al. 1997, 1999, Maddux et al. 2001) reported the role of oxidative stress in the pathogenesis of insulin resistance. A decline in reduced glutathione, vitamin C, vitamin E, and lipoic acid is noted in patients with diabetes (Shigeta et al. 1961, Maxwell et al. 1997, Opara et al. 1999). In vitro studies and studies in animal insulin resistance models have shown that antioxidants, especially α-lipoic acid, vitamin E, and vitamin C improve insulin sensitivity (Evans & Goldfine 2000, Jacob et al. 2000). When rat L6 muscle cells and mouse 3T3L1 adipocytes were exposed to oxidative stress, insulin-stimulated glucose uptake was inhibited (Rudich et al. 1999, Maddux et al. 2001). Antioxidant treatment to these cell lines protects against the oxidative stress-induced insulin resistance (Maddux et al. 2001, Evans et al. 2002). However, the outcomes of several clinical studies have yielded disappointing results when these antioxidants were used independently (Yusuf et al. 2000). Studies have shown that when these antioxidants were used in combination their insulin-sensitizing/antidiabetic action was enhanced through their interaction and effective regeneration of endogenous antioxidants (Evans et al. 2002). Although the insulin-sensitizing property of antioxidants is identified, the potential mechanism(s) by which antioxidants improve insulin sensitivity in insulin resistance is not clearly known.

Elevated levels of fatty acids are known to cause the production of reactive oxygen species (ROS) and oxidative stress (Carisson et al. 1999, Rao & Reddy 2001). Many studies have shown that the ROS induces activation of multiple stress-sensitive serine kinase cascades and their role in the pathogenesis of insulin resistance (Adler et al. 1999, Qiao et al. 1999). One such major intracellular serine kinase target for oxidative stress is C-Jun-N-terminal kinase (JNK). The JNK pathway is the member of serine/threonine protein kinase superfamily of mitogen-activated protein kinase pathway. The JNK pathway is activated by variety of exogenous and endogenous stress signals including oxidative stress, osmotic stress, proinflammatory cytokines, heat shock, and u.v. irradiation (Tibbles & Woodgett 1999). The activation of JNK pathway is known to interfere with insulin action (Agirre et al. 2000). It has been reported that JNK pathway is activated in various tissues under diabetic condition (Purves et al. 2001, Hirosumi et al. 2002, Qiao et al. 1999). Thus, it is likely that the oxidative stress-induced activation of JNK pathway is a crucial mediator of the progression in insulin resistance. Even though oxidative stress, the activation of JNK pathway and impaired insulin signaling is reported in high-fat feeding, their associated role in the pathogenesis of insulin resistance and effect of antioxidants are not studied in detail. It is worth investigating the interrelationship between the above-mentioned biochemical events because these studies could yield new insights into the molecular basis of obesity-/dyslipidemia-induced insulin resistance as well as help to identify new pharmaceutical targets for the treatment of type 2 diabetes. In view of the above, the present study was designed to investigate the effect of antioxidants on redox balance, insulin action, and JNK pathway in high-fat-fed rats which closely mimic the obesity-/dyslipidemia-induced insulin resistance in humans (Buettner et al. 2007).

Materials and Methods

Materials

Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), penicillin, streptomycin, glucose oxidase, 1,1-diphenyl-2-picryl hydrazyl (DPPH), insulin (human recombinant), sodium vandate, phenyl methyl sulfonyl fluoride, aprotinin, leupeptin, okadaic acid, and all other chemicals were purchased from Sigma Chemicals. 2-Deoxy-14C-d-glucose was purchased from Amersham Life Sciences. Insulin receptor, IRS-1, JNK1, phosphor-JNK1, and phosphor-tyrosine antibodies were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Phosphor-serine antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein A-agarose slurry was purchased from Bangalore Genei (Bangalore, India).

Treatment of animals

Male Wistar rats weighing 150–200 g were maintained in the standard laboratory conditions at 22±2 °C with 12-h darkness:12-h light cycle. All experimental procedures were approved by the Institute Animal Experimentation Ethics Committee. The rats were randomly assigned to the following groups.

  1. Control – rats fed with rodent chow.

  2. Control+Antioxidants – rats fed with rodent chow supplemented with 0.2% (w/w) vitamin E, 0.3% (w/w) vitamin C, and 0.5% (w/w) α-lipoic acid.

  3. High-fat diet – Rats fed with high-fat diet.

  4. High-fat diet+Antioxidants – Rats fed with high-fat diet supplemented with 0.2% (w/w) vitamin E, 0.3% (w/w) vitamin C, and 0.5% (w/w) α-lipoic acid.

The semi-purified high-fat diet was prepared as described previously (Storlien et al. 1986), with 59% of total calories derived from fat, 21% from protein, and 20% from carbohydrate. The energy of the high-fat diet was 5.2 kcal/g whereas that of the chow diet was 3.3 kcal/g. The rats were provided with specific diets and water ad libitum for 9 weeks.

i.p. Glucose and insulin tolerance tests in male Wistar rats

At the end of the 9-week experimental period, i.p. glucose and insulin tolerance tests were performed as described by Yuan et al. (2001). i.p. Glucose tolerance test was performed in overnight fasted rats after i.p. injection of glucose (2.0 g/kg body weight). For insulin tolerance test, insulin was injected i.p. (2.0 U/kg body weight) after 6-h fasting. Blood samples were taken at different time intervals to estimate plasma glucose. Area under curve was calculated from the graph using NCSS software (NCCS2007 for Windows, Statistical solutions, CA, USA).

Analysis of plasma parameters

After 9 weeks of fat feeding, plasma glucose, triglycerides, and total cholesterol were estimated in overnight fasting samples using standard reagent kits in 550 Express plus autoanalyzer (Ciba Corning Diagnostics, MA, USA). Fasting plasma insulin was estimated using rat insulin ELISA kit purchased from Boehringer Mannheim. Homeostatic model assessment-insulin resistance (HOMA-IR) was calculated using the following formula: HOMA-IR=fasting insulin (μU/ml)×fasting glucose (mM)/22.5 (Pickavance & Wilding 2007).

Cell culture

Rat L6 myoblasts (American Type Culture Collection, Manassas, VA, USA) were cultured (37 °C, 5% CO2) in growth medium (DMEM, 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin). L6 myoblasts were allowed to differentiate into myotubes in differentiation medium (DMEM, 2% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin) as described previously (Vinayagamoorthi et al. 2006).

Hydrogen peroxide treatment to rat L6 myotubes

Rat L6 myotubes were incubated with antioxidant mixture (5 μM vitamin E, 300 μM lipoic acid, and 500 μM vitamin C) in DMEM supplemented with 0.5% BSA for 18 h. After antioxidant treatment, myotubes were washed with DMEM supplemented with 0.5% BSA and incubated in DMEM (phenol red free) supplemented with 0.5% BSA, 25 mU/ml glucose oxidase, and 5 mM glucose for 12 h. H2O2 generated by this system was estimated from the media collected at different time intervals using Amplex red – hydrogen peroxide/peroxidase assay kit (Molecular Probes, NJ, USA). Creatine kinase (CK) activity was measured in the culture medium using colorimetric kit (Teco Diagnostics, USA) in 550 Express plus autoanalyzer (Ciba Corning Diagnostics). H2O2-induced cytotoxicity was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described previously (Ferrai et al. 1999).

Determination of glucose transport into rat L6 myotubes

Rat L6 myotubes were treated with antioxidants (18 h) followed by H2O2 (12 h) as described above. After glucose/glucose oxidase treatment, L6 myotubes were washed with DMEM supplemented with 0.5% BSA and then incubated with serum-free DMEM for 3 h. After serum starvation, insulin (100 nM) was added to the serum-free medium and further incubated for 30 min. Myotubes were washed with HBS (140 nM NaCl, 20 mM HEPES pH 7.4, 5.0 mM KCl, 2.5 nM MgSO4, and 1.0 mM CaCl2) and glucose uptake was measured as described previously (Blair et al. 1999). Briefly, after insulin stimulation, myotubes were incubated with 2-deoxy-14C-d-glucose (0.5 μCi/ml) in HBS for 10 min. Radioactive medium was aspirated rapidly and the cells were washed with ice-cold isotonic saline (0.9% NaCl). The cells were lysed in 0.05 M NaOH, and radioactivity was determined by liquid scintillation counting (Packyard Top Count LSC, GMI Inc., MN, USA). Protein content of lysates was estimated by the method of Bradford (1976). Non-specific glucose uptake was determined in the presence of cytochalasin B (50 μM) an inhibitor of facilitative glucose transport and was subtracted from total uptake.

Analysis of oxidative stress parameters and antioxidant status in fat-fed rats and rat L6 myotubes

At the end of 9-week experimental period, plasma and skeletal muscle malondialdehyde (MDA) were estimated by TBARS method (Yagi 1984), and erythrocyte-reduced glutathione content was determined using Ellman's reagent (5,5′-dithio-bis-2-nitrobenzoic acid) as described by Beutle et al. (1963). Hemoglobin content of blood was estimated by cyanmethemoglobin method of Drabkin & Austin (1932) and the catalase activity in erythrocytes was estimated by the method of Aebi (1984).

Rat L6 myotubes were treated with LA (18 h) followed by H2O2 (12 h) as described previously and lysed in 0.5 ml PBS by repeated freezing and thawing. Total antioxidant status of cell lysates was quantified by trolox equivalent antioxidant capacity assay using DPPH as described previously (Vinayagamoorthi et al. 2006). Reduced glutathione concentration in cell lysates was estimated as described by Beutle et al. (1963).

Insulin stimulation

At the end of 9-week experimental period, the animals were killed with and without insulin stimulation. Insulin stimulation was performed as described earlier by i.p. injection of 15 units of insulin/kg body weight (Youngren et al. 2001). After an interval of 30 min for the maximum effect of insulin to occur, the animals were killed and the hind limb muscle was removed. Muscle homogenates were prepared in homogenization buffer (50 mM Tris–HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM sodium vandate, 1 mM phenyl methyl sulfonyl fluoride, 1 mM aprotinin, 1 mM leupeptin, and 0.5 μg/ml okadaic acid) as described previously (Saad et al. 1992).

Rat L6 myotubes were treated with antioxidants (18 h) followed by H2O2 (12 h) as described previously and incubated with serum-free DMEM for 30 min. After serum starvation, insulin (100 nM) was added to the serum-free medium and incubated for 15 min. Myotubes were washed with ice-cold PBS and scraped into ice-cold homogenization buffer. Muscle homogenates and cell lysates were centrifuged at 12 000 g for 15 min at 4 °C. Protein content of the supernatant collected was estimated by the method of Bradford (1976).

Western blotting analysis of JNK pathway

Muscle homogenates and rat L6 cell lysates (100 μg protein) were resolved by 12% SDS-PAGE, electrotransferred onto nitrocellulose membrane, and immunoblotted with antibody specific to phosphorylated JNK1. Furthermore, the membrane was stripped of bound antibodies and then reprobed with antibody specific to JNK1. Protein bands were visualized by enhanced chemiluminescence method using Amersham ECL kit (Amersham Life Sciences). Bands were scanned using a densitometer (Bio-Rad, Model GS-710) and quantified by Quantity 1 software (Bio-Rad). The band density of phosphorylated JNK1 was normalized with the total JNK1.

Insulin signaling analysis

Muscle homogenates (250 μg protein) and rat L6 cell lysates (500 μg protein) were incubated overnight at 4 °C with insulin receptor and IRS-1 antibodies. The immune complex was captured by adding 50 μl protein A-agarose beads for 2 h at 4 °C. The immune complex was pelleted at 12 000 g for 15 min at 4 °C and washed thrice with homogenization buffer. The immune complex was suspended in Laemmli (1970) sample buffer and boiled for 5 min. Protein A agarose was removed from the denatured proteins by centrifugation at 12 000 g for 15 min at 4 °C. The supernatant was resolved by 8.0% SDS-PAGE and further electrotransferred to nitrocellulose membrane. Proteins were immunoblotted with the antibody specific for phosphorylated tyrosine, and the immunoblot was stripped of bound antibodies and then reprobed with an antibody specific to phosphorylated serine. Protein band detection and quantification were performed as mentioned previously. The band densities of tyrosine and serine phosphorylation were normalized with the band densities of immunoprecipitated insulin receptor and IRS-1 respectively.

RT-PCR analysis of antioxidant enzymes in male Wistar rats

At the end of 9-week experimental period, RT-PCR analysis was performed in skeletal muscle of male Wistar rats as described previously (Piro et al. 2002, Bhor et al. 2004). Briefly, total RNA was extracted from rat skeletal muscle using Trizol reagent (Invitrogen). The cDNA was synthesized from total RNA (2 μg) using cDNA synthesis kit (Roche Molecular Biochemicals) following the manufacturer's protocol. The PCR was performed using 2 μl cDNA in a total volume of 20 μl reaction. Amplification of all the genes of interest was done with the forward and reverse gene-specific primers (Bhor et al. 2004) at a concentration of 0.6 μM in PCR mixture (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris–HCl (pH 9.0), 200 μM each dNTP, 2.5U Taq polymerase). Reverse transcription and gene amplification reactions were carried out in a gradient thermal cycler (Eppendorff, Gradient Mastercycler, Germany). The electrophoresis analysis of PCR products was performed in 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. The gels were photographed using a gel-documentation system (Bio-Rad, Gel Doc EQ) and were analyzed in Quantity 1 software (Bio-Rad).

Statistical analysis

Data are expressed as mean±s.e.m. Analysis of difference between the means of groups was done by one-way ANOVA followed by Tukey's post-test. A P value <0.05 was considered statistically significant.

Results

Antioxidants improve insulin sensitivity in fat-fed rats and H2O2-treated L6 myotubes

Glucose and insulin tolerance tests (Fig. 1(a) and (b)) in male Wistar rats showed that fat feeding for 9 weeks increases the area under curve significantly (P<0.01) compared with control and antioxidant-supplemented groups. The fasting insulin and HOMA-IR (Table 1) were significantly (P<0.001) increased in fat-fed group compared with other groups. Increased area under curve and HOMA-IR in fat-fed group show impaired glucose tolerance and insulin resistance. Antioxidant supplementation along with high-fat diet improves the glucose tolerance and insulin sensitivity in fat-fed rats.

Figure 1

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Figure 1

Antioxidants improve insulin sensitivity in fat-fed rats and H2O2-treated rat L6 myotubes: male Wistar rats were fed with specific diet for 9 weeks (filled square, control (n=9); filled diamond control+antioxidants (n=10); filled triangle high fat diet (HFD; n=8); X HFD+antioxidants (AO; n=8)). At the end of 9 weeks of treatment, (a) i.p. glucose tolerance test and (b) i.p. insulin tolerance test were performed as described in Materials and Methods. Data are expressed as means with their standard errors indicated by vertical bars. Area under curve (AUC) of different groups calculated from the graph using NCCS software. The AUC for the glucose tolerance test of control, control+antioxidants, HFD, and HFD+antioxidants was 15 087±894, 14 543±702, 22 333±1453*, and 16 108±769 respectively. The AUC for the insulin tolerance test of control, control+antioxidants, HFD, and HFD+antioxidants was 4352±239, 4416±227, 5816±263*, and 4442±232 respectively.*P<0.01 compared with control and antioxidant-treated groups. (c) Rat L6 myotubes were pre-treated with antioxidants for 18 h and then exposed to H2O2 for 12 h. 2-Deoxy-14C-d-glucose uptake was measured in myotubes after insulin stimulation as described in the Materials and Methods. Data represent mean±s.e.m. of three independent experiments done in triplicate. *P<0.001 in comparison with basal glucose uptake.

Citation: Journal of Endocrinology 197, 2; 10.1677/JOE-08-0061

Table 1

Effect of high-fat diet and antioxidants on biochemical and oxidative stress parameters. Male Wistar rats were fed with specific diet for 9 weeks. After 9 weeks of treatment, blood parameters were estimated in overnight fasting sample. Results shown are mean±s.e.m.

ParameterControl (n=9)Control+AO (n=10)HFD (n=8)HFD+AO (n=8)
S. no.
1Glucose (mg/dl)84.5±15.384.9±12.981.62±7.390.25±11.5
2Insulin (μU/ml)21.03±2.5722.32±3.1843.23±4.4925.43±3.97
3HOMA-IR4.2±0.64.6±1.058.6±1.25.6±1.3
4Total cholesterol (mg/dl)68.11±9.163.4±9.083.3±13.2*85.2±12.3*
5Triglycerides (mg/dl)69.33±7.168.8±6.5111.1±12.9107.5±12.0
6Body weight (g)272.6±9.8282.5±8.8331.0±12.4324.0±28
7Epididymal fat weight (g)1.31±0.231.242±0.273.03±0.323.18±0.54
8Plasma TBARS (micromols/l)2.88±0.482.83±0.444.10±0.313.01±0.60
9Muscle TBARS (micromols/mg protein)11.86±5.311.52±6.521.15±6.110.86±5.4
10Erythrocyte-reduced glutathione (mg/g Hb)4.74±0.454.76±0.782.88±0.394.36±0.67
11Erythrocyte catalase (K/ml)8.34±1.466.16±0.113.51±2.287.03±2.16

*P<0.05 compared with control, P<0.001 compared with control. HFD, high-fat diet, AO, antioxidants.

Addition of 25 mU/ml glucose oxidase, 5mM glucose for 12 h resulted in a H2O2 concentration in the medium that achieved a steady state of 40±10 μM after 30 min. When cells were incubated at this concentration of glucose oxidase, basal uptake was not affected, whereas insulin-stimulated glucose uptake was completely abolished (Fig. 1(c)). Pre-treatment with antioxidants had no effect on basal glucose transport, but restored insulin-stimulated glucose uptake in cells exposed to oxidative stress.

Effect of high-fat diet and antioxidants on plasma lipids, body weight, and epididymal fat weight

As shown in Table 1, fat feeding for 9 weeks in male Wistar rats significantly increases the fasting plasma triglycerides (P<0.001), total cholesterol (P<0.01), body weight, and epididymal fat weight (P<0.001) compared with control group. This reveals induction of insulin resistance by high-fat feeding in rats closely mimics the obesity-/dyslipidemia-induced insulin resistance in humans. Antioxidant supplementation along with high fat did not have any significant effect on the above parameters.

Antioxidants inhibit IRS-1 serine phosphorylation and improve insulin-stimulated IRS-1 tyrosine phosphorylation in fat-fed rats and H2O2-treated rat L6 myotubes

Fat feeding to rats and H2O2 treatment to L6 myotubes did not have significant effect on insulin receptor tyrosine and serine phosphorylation (data not shown). However, feeding rats with high-fat diet and H2O2 treatment to L6 myotubes significantly (P<0.01) reduced the insulin-stimulated tyrosine phosphorylation of IRS-1 compared with control and antioxidant-treated groups (Fig. 2(a) and (c)). In addition, above treatments significantly (P<0.01) increased the basal serine phosphorylation of IRS-1 compared with other groups (Fig. 2(b) and (d)). Antioxidant treatment inhibited the high-fat diet and H2O2-induced IRS-1 serine phosphorylation and improved the insulin-stimulated IRS-1 tyrosine phosphorylation.

Figure 2

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Figure 2

Antioxidants inhibit IRS-1 serine phosphorylation and preserve insulin-stimulated IRS-1 tyrosine phosphorylation. Insulin signaling was investigated by western blotting and immunoprecipitation analysis in male Wistar rat skeletal muscle homogenates and rat L6 myotube lysates as described in Materials and Methods. (a) Immunoprecipitation and immunoblotting analysis of IRS-1 tyrosine phosphorylation in male Wistar rats. (b) Immunoprecipitation and immunoblotting analysis of IRS-1 serine phosphorylation in male Wistar rats. (c) Immunoprecipitation and immunoblotting analysis of IRS-1 tyrosine phosphorylation in rat L6 myotubes. (d) Immunoprecipitation and immunoblotting analysis of IRS-1 serine phosphorylation in rat L6 myotubes. A representative immunoblot of three independent experiments is shown. Results shown are mean±s.e.m. of three experiments. C=control, C+A=control+antioxidant, H=HFD, H+A=HFD+antioxidant, and AO=antioxidant pre-treatment. *P<0.001 compared with basal. P<0.001 compared with insulin-stimulated control and insulin-stimulated antioxidant groups. $P<0.01 compared with control and antioxidant groups.

Citation: Journal of Endocrinology 197, 2; 10.1677/JOE-08-0061

Figure 3

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Figure 3

Antioxidants inhibit JNK pathway in fat-fed rats and H2O2-treated rat L6 myotubes. Western blotting analysis of JNK pathway was performed in (a) male Wistar rat skeletal muscle homogenates and (b) rat L6 myotube lysates as described in Materials and Methods. A representative immunoblot of three independent experiments is shown. Results shown are mean±s.e.m. of three experiments. C=control, C+A=control+antioxidant, H=HFD, H+A=HFD+antioxidant, and AO=antioxidant pre-treatment. *P<0.001 compared with control and antioxidant groups.

Citation: Journal of Endocrinology 197, 2; 10.1677/JOE-08-0061

Antioxidants restore redox balance and inhibit redox-sensitive JNK pathway in fat-fed rats and H2O2-treated rat L6 myotubes

Fat feeding to rats for 9 weeks significantly (P<0.001) increased the plasma and skeletal muscle TBARS (Table 1) and expression of skeletal muscle antioxidant enzymes (Fig. 4(a)) compared with control and antioxidant groups. High-fat feeding significantly (P<0.001) increased the erythrocyte catalase activity and decreased the reduced glutathione concentration (Table 1) compared with control and antioxidant groups. High-fat diet also activated the redox-sensitive JNK pathway in skeletal muscle (Fig. 3(a)). Antioxidant supplementation preserved the redox balance in fat-fed rats and inhibited the activation of JNK pathway.

Figure 4

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Figure 4

Antioxidants preserve the redox balance in fat-fed rats and H2O2-treated rat L6 myotubes. (a) RT-PCR analysis of antioxidant enzymes in skeletal muscle mRNA of male Wistar rats. A representative gel picture of four independent experiments is shown. Results shown are mean±s.e.m. of four experiments. C=control, C+A=control+antioxidant, H=HFD, H+A=HFD+antioxidant. *P<0.001 compared with control and antioxidant groups. (b) Total antioxidant status in cell lysates of rat L6 myotubes. (c) Reduced glutathione levels in cell lysates of rat L6 myotubes. AO = antioxidant pre-treatment. Data represent mean±s.e.m. of three independent experiments done in triplicate.*P<0.01 compared with control and antioxidant-treated cells. GPX, glutathione peroxidase; GSH, glutathione; TEAC, Trolox equivalent antioxidant capacity.

Citation: Journal of Endocrinology 197, 2; 10.1677/JOE-08-0061

H2O2 treatment to L6 cells significantly (P<0.01) decreased the total antioxidant capacity (Fig. 4(b)), decreased the reduced glutathione levels (Fig. 4(c)), and activated the redox-sensitive serine kinase JNK pathway (Fig. 3(b)). Pre-treatment with antioxidant restored the redox balance and inhibited the oxidative stress-induced activation of JNK pathway. To determine whether H2O2 treatment causes cell toxicity, we performed the cytotoxicity (MTT) assay and measured the release of skeletal muscle cytosolic marker enzyme creatine kinase (CK) in culture medium after incubation with glucose oxidase. The MTT assay and CK activity in the culture medium did not show any significant difference between the groups (data not shown). Thus, under the experimental conditions of the present study, the effect of antioxidants is due to the protection against the oxidative stress and not due to simply protecting the cells from cell death.

Discussion

In the present study, we have investigated the effect of antioxidants on insulin sensitivity, proximal insulin signaling, redox balance, and JNK pathway in fat-fed rats and cultured rat L6 myotubes. Fat feeding to rats for 9 weeks impaired insulin sensitivity, redox balance, and insulin-stimulated IRS-1 tyrosine phosphorylation. High-fat diet significantly increased the IRS-1 serine phosphorylation and activated JNK pathway. When we exposed rat L6 myotubes to oxidative stress using a H2O2 generating system, the insulin-stimulated glucose transport and IRS-1 tyrosine phosphorylation were decreased. Oxidative stress also increased the IRS-1 serine phosphorylation, impaired intracellular redox balance, and activated the redox-sensitive serine kinase – JNK pathway. Antioxidant treatment restored redox balance, inhibited JNK activation and IRS-1 serine phosphorylation, preserved insulin-stimulated IRS-1 tyrosine phosphorylation, and improved insulin sensitivity in both fat-fed rats and H2O2-treated rat L6 myotubes.

Previous studies in high-fat-fed rats demonstrated severe insulin resistance and defects in proximal insulin signaling pathway (Saad et al. 1992). Hansen et al. (1998) reported a decreased insulin-stimulated IRS-1 tyrosine phosphorylation in fat-fed rats. In the present study, fat feeding for 9 weeks significantly increased the IRS-1 serine phosphorylation and decreased the insulin-stimulated IRS-1 tyrosine phosphorylation. Numerous studies have documented increased IRS-1 serine phosphorylation as a potential molecular mechanism for insulin resistance (Le Roith & Zick 2001, Sykiotis & Papavassiou 2001, Anna et al. 2005). In patients with type 2 diabetes and animal models of insulin resistance, IRS-1 serine phosphorylation was found to be increased (Qiao et al. 1999). IRS-1 contains a total of 232 serine/threonine amino acid residues that provide a potential site for phosphorylation by Ser/Thr kinases (Werner et al. 2004). Serine phosphorylation of IRS-1 impairs its interaction with the juxtamembrane domain of insulin receptor and thus renders IRS-1 as a poorer substrate for insulin receptor kinase (Paz et al. 1997). Numerous agents that induce insulin resistance, such as TNF-α, okadaic acid, platelet-derived growth factor, and angiotensin II, increase IRS-1 phospho-serine content (Sykiotis & Papavassiou 2001). Basal levels of IRS-1 serine phosphorylation are increased in cells under various conditions, leading to observable shifts in electrophoretic mobility (Tanti et al. 1994, Mothe & Obberghen 1996). The magnitudes of the shifts demonstrate that multiple sites are phosphorylated. Numerous studies have shown that serine phosphorylation of IRS-1 (in mouse) at ser302, ser307, ser612, ser636, ser731, and ser789 inhibits its interaction with insulin receptor (Mothe & Obberghen 1996). Out of all this, the inhibitory effect of IRS-1 ser307 phosphorylation on insulin action is extensively studied (Tanti et al. 1994, Mothe & Obberghen 1996, Paz et al. 1997, Sykiotis & Papavassiou 2001). However, there are reports for the requirement of clusters of serine phosphorylation to disturb IRS-1 function (Werner et al. 2004). To this end, it is evident that different stimuli may phosphorylate different serine residues of IRS-1 and inhibit insulin action. Thus, the specific signals and the phosphorylated serine residues, which inhibit IRS-1 function, need to be identified. In the present study, we identified the oxidative stress-induced increased global serine phosphorylation of IRS-1 and impaired insulin action in fat-fed rats and cultured rat L6 myotubes. However, additional studies are clearly required to identify the oxidative stress induced specific phosphorylated serine residues and their role in insulin action.

Increased intake of fat and its derivatives are reported to cause oxidative stress (Bakker et al. 2000). Elevated fats can cause oxidative stress due to increased β-oxidation (Otczalk & Schonfeld 1993, Carisson et al. 1999) and mitochondrial respiration (Rao & Reddy 2001). In the process of mitochondrial respiration, molecular oxygen is essential for the production of ATP. During the course of normal oxidative phosphorylation between 0.4 and 4.0% of the total oxygen consumed is converted into the free radical superoxide anion (Chance et al. 1979, Boveris 1984). Subsequently, can be converted into other ROS. However, the endogenous antioxidant system that exists within the cells neutralize these ROS and maintains redox balance. Oxidative stress sets in when endogenous antioxidant network fails to provide a compensatory response to restore cellular redox balance. In agreement with previous studies (Rudich et al. 1997,1999, Maddux et al. 2001), our results from cultured rat L6 myotubes demonstrate oxidative stress can cause insulin resistance. Recently we reported the reduction in insulin-stimulated glucose uptake in rat L6 muscle cell lines when exposed to oxidative stress and its reversal on vitamin E treatment (Vinayagamoorthi et al. 2006). ROS can function as signaling molecules and activate a number of redox-sensitive Ser/Thr kinase cascades linked to insulin resistance (Evans et al. 2002). Our results show that fat feeding to rats impairs redox balance and activates the redox-sensitive serine kinase – JNK pathway. Evidence from cellular models and transgenic animals demonstrated the role of JNK in the pathogenesis of insulin resistance. Support for the importance of JNK pathway in insulin resistance is provided by the results of gene knockout experiments in mice. Suppression of JNK pathway improves insulin sensitivity in db/db mice and sucrose-fed rats (Nakatani et al. 2004). Several studies have shown inhibitors of JNK pathway improve insulin signaling and insulin sensitivity (Kaneto 2005, Liu & Rondinone 2005). Agirre et al. (2000) showed in CHO cells, that anisomycin a strong activator of JNK, stimulated the binding activity of JNK to IRS-1, and inhibited the insulin-stimulated tyrosine phosphorylation of IRS-1.

Our results from rat L6 myotubes demonstrate that oxidative stress-induced insulin resistance is associated with the activation of JNK pathway and increased IRS-1 serine phosphorylation. Antioxidant pre-treatment to rat L6 myotubes preserved redox balance, inhibited JNK pathway and IRS-1 serine phosphorylation, retained insulin-stimulated IRS-1 tyrosine phosphorylation, and restored insulin sensitivity. In this regard, here we propose (Fig. 5) that fat feeding to rats increases ROS formation and causes redox imbalance. This results in the activation of redox-sensitive JNK cascade. Oxidative stress-induced activation of JNK in turn phosphorylates IRS-1 and increases phosphor-serine content of IRS-1. Increased IRS-1 serine phosphorylation inhibits its interaction with insulin receptor and decreases insulin-stimulated IRS-1 tyrosine phosphorylation. This results in insulin resistance in fat-fed rats. The insulin-sensitizing property of antioxidants in high-fat-induced insulin resistance could be related to their ability to preserve the intracellular redox balance and thereby preventing the activation of redox-sensitive JNK pathway and IRS-1 serine phosphorylation. In conclusion, the present study helps us to understand the molecular basis of high-fat-induced insulin resistance and insulin-sensitizing property of antioxidants. More detailed understanding of the role of oxidative stress and antioxidants on redox-sensitive Ser/Thr kinase cascades in humans and animal models of insulin resistance might open novel therapeutic targets for the treatment of type 2 diabetes and its complications.

Figure 5

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Figure 5

Proposed mechanism for insulin resistance and beneficial effect of antioxidants in high-fat-fed rats.

Citation: Journal of Endocrinology 197, 2; 10.1677/JOE-08-0061

Acknowledgements

We are grateful to Indian Council of Medical Research (ICMR) for providing senior research fellowship to R V. The authors do not have conflict of interest in this work.

References

  • AdlerVYinZTewKDRonaiZ1999Role of redox potential and reactive oxygen species in stress signaling. Oncogene1861046111.

  • AebiH1984Catalase in vitro. Methods in Enzymology105121126.

  • AgirreVUchiudaTYenushLDavisRWhiteMF2000The c-Jun NH(2) terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser (307). Journal of Biological Chemistry2773001030018.

  • AnnaDAnitaOFredrikHNPeterS2005Attenuation of insulin – stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes. Journal of Biological Chemistry2803438934392.

  • BakkerSJIjzermenRGTeerlinkTWesterhoffHVGansROHeineRJ2000Cytosolic triglycerides and oxidative stress in central obesity: the missing link between excessive atherosclerosis, endothelial dysfunction, and β-cell failure?Atherosclerosis1481721.

  • BeutleEDuronOKellyBM1963Improved method for the determination of blood glutathione. Journal of Laboratory and Clinical Medicine61882888.

  • BhorVMRaghuramNSivakamiS2004Oxidative damage and altered antioxidant enzyme activities in the small intestine of streptozotocin-induced diabetic rats. International Journal of Biochemistry and Cell Biology368997.

  • BlairASHajduchELitherlandGJHundalSH1999Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress. Journal of Biological Chemistry2743629336299.

  • BoverisA1984Determination of the production of superoxide radicals and hydrogen peroxide in mitochodria. Methods in Enzymology105429435.

  • BradfordM1976A rapid and sensitive method for the quantification of microgram quantity of protein utilizing the principle of protein dye binding. Analytical Biochemistry72248254.

  • BuettnerRScholmerichBBollheimerC2007High-fat diets: modeling the metabolic disorders of human obesity in rodents. Obesity15798808.

  • CarissonGBorgLAWelshN1999Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rats pancreatic islets in vitro. Endocrinology14034223428.

  • ChanceBSiesHBoverisA1979Hydrogen peroxide metabolism in mammalian organs. Physiological Reviews59527605.

  • DrabkinDLAustinJN1932Spectrophotometric constants for common hemoglobin derivatives in human, dog and rabbit. Journal of Biological Chemistry98719733.

  • EvansJLGoldfineID2000α-Lipoic acid: a multi-functional antioxidant that improves insulin sensitivity in patients with type 2 diabetes. Diabetes Technology and Therapeutics2401413.

  • EvansJLGoldfineIDMadduxBAGrodskyGM2002Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocrine Reviews23599622.

  • FerraiMFornasieroMCIsettaAM1999MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. Journal of Immunological Methods131165172.

  • HansenPAHanHDMarshallBANolteLAChenMMMuecklerMHolloszyOJ1998A high fat diet impairs stimulation of glucose transport in muscle. Functional evaluation of potential mechanisms. Journal of Biological Chemistry2732615726163.

  • HirosumiJTunemanGChangLGorgunCZUysalKTMaedaKKarinMHotamisligilGS2002A central role for JNK in obesity and insulin resistance. Nature420333336.

  • JacobSLehmannRRettKHaringHU2000Oxidative stress and insulin action: a role for antioxidant. In Antioxidant in Diabetes Management pp 319338. Eds PackerLRosenPTritschlerHKingGL. New York: Marcel dekker.

  • KanetoH2005The JNK pathway as a therapeutic target for diabetes. Expert Opinion on Therapeutic Targets9581592.

  • KrssakMPetersenKFDresnerA1999Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: H-1 NMR spectroscopy study. Diabetes42113116.

  • LaemmliUK1970Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature227680685.

  • LengYKarlssonHKZierathJR2004Insulin signaling defects in type 2 diabetes. Reviews in Endocrine and Metabolic Disorders5111117.

  • LiuGRondinoneCM2005JNK bridging the insulin signaling and inflammatory pathway. Current Opinion in Investigational Drugs6979987.

  • MadduxBASeeWLawrenceJCJrGoldfineALGoldfineIDEvansJL2001Production against oxidative stress induced insulin resistance in rat L6 muscle cells by micromolar concentrations of α-lipoic acid. Diabetes50404410.

  • MaxwellSRJThomansonHSandlerDLeguenCBaxterMAThorpeGHJonesAFBarnettAH1997Antioxidant status in patients with uncomplicated insulin-dependent and non -insulin dependent diabetes mellitus. European Jouranl of Clinical Investigation27487490.

  • MotheIObberghenVE1996Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action. Journal of Biological Chemistry2711122211227.

  • NakataniYKanetoHKawamoriDHatazakiMMiyatsukaTMatsuokaTAKajimotoYMatsuhisaMYamasakiYHoriM2004Modulation of the JNK pathway in liver affects insulin resistance status. Journal of Biological Chemistry2794580345809.

  • OparaECAbdel RahmanESolimanEKamelWASoukaSLoweJEAbdel-AleemS1999Depletion of total antioxidant capacity in type 2 diabetes. Metabolism4814141417.

  • OtczalkLSchonfeldP1993Effect of fatty acids on energy coupling processes in mitochondria. Biochemica et Biophysica Acta11834157.

  • PanDALilliojaSKriketosADMilnerMRBaurLRBogardusCJenkinsABStorlienLH1997Skeletal muscle triglyceride levels are inversely related to insulin action. Diabetes46983988.

  • PazKHemiRLeRoithDKarasikAElhananyEKanetyHZiokY1997A molecular basis of insulin resistance: elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the jextamembrane region of the insulin receptor and impairs their ability to undergo insulin induced tyrosine phosphorylation. Journal of Biological Chemistry2722991129918.

  • PerseghinGGhoshSGerowKShulmanGI1997Metabolic defects in lean nondiabetic offspring of NIDDM parents: a cross-sectional study. Diabetes4610011009.

  • PhillipsDICaddySIIicVFieldingBAFraynKNBorthwickACTaylorR1996Intramuscular triglycerides and muscle insulin sensitivity: evidence for a relationship in nondiabetic subjects. Metabolism45947950.

  • PickavanceLCWildingJP2007Effects of S 15511, a therapeutic metabolite of insulin sensitizing agent S15261, in Zucker Diabetic Fatty rat. Diabetes Obesity and Metabolism9114120.

  • PiroSAnelloMDi PietroCLizzioMNPatane`GRabuazzoAMVigneriRPurelloMPurelloF2002Chronic exposure to fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. Metabolism5113401347.

  • PurvesTMiddlemasAAgthongSJudeEBBoulronAJFeroyhoughPTomlinsonDR2001A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy. FASEB Journal1525082514.

  • QiaoLYGoldbergJLRussellJCSunXJ1999Identification of enhanced serine kinase activity in insulin resistance. Journal of Biological Chemistry2741062510632.

  • RaoMSReddyJK2001Peroxisomal β-oxidation and steatohepatitis. Seminars in Liver Disease214355.

  • LeRoithDZickY2001Recent advances in our understanding of insulin action and insulin resistance. Diabetes Care24588597.

  • RudichAKozlovskyNPotashnikRBashanN1997Oxidant stress reduces insulin responsiveness in 3T3LI adipocytes. American Journal of Physiology35E935E940.

  • RudichATiroshAPotashnikRKhamaisiMBashanN1999Lipoic acid protects against oxidative stress induced insulin resistance impairment in insulin stimulation of proteinkinase B and glucose transport in 3T3L1 adipocytes. Diabetologia42949957.

  • SaadMJAArakiEMiralpeixMRothenbergPLWhiteMFKohnCR1992Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance. Journal of Clinical Investigation9018391849.

  • SaltielARKahnCR2001Insulin signaling and the regulation of glucose and lipid metabolism. Nature414799806.

  • ShigetaYHiraizumiGWadaMOjiKYoshidaT1961Study on the serum levels of thioctic acid in patients with various diseases. Journal of Vitaminology74852.

  • SpiegelmanBMFlierJS2001Obesity and the regulation of energy balance. Cell104531543.

  • StorlienLHJamesDEBurleighKMChisholmDJKraegenEW1986Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats. American Journal of Physiology. Endocrinology and Metabolism251E576E583.

  • SykiotisGPPapavassiouAG2001Serine phosphorylation of insulin receptor substrate -1: a novel target for the reversal of insulin resistance. Molecular Endocrinology1518641869.

  • TantiJFGremeauxTObberghenEMarchand-BrustelY1994Serine/threonine phosphorylation of insulin receptor substrate 1 modulates insulin receptor signaling. Journal of Biological Chemistry26960516057.

  • TibblesLAWoodgettJR1999The stress activated protein kinase pathways. Cellular and Molecular Life Sciences 5512301254.

  • VinayagamoorthiRBobbyZSelvarajNSridharMG2006Vitamin E protects the insulin sensitivity and redox balance in rat L6 muscle cells exposed to oxidative stress. Clinica Chimica Acta367132136.

  • WernerEDLeeJHansenLYuanMShoelsonSE2004Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302. Journal of Biological Chemistry2793529835305.

  • YagiK1984Assay for blood plasma or serum lipid peroxides. Methods in Enzymology1052831.

  • YoungrenJFJohnPBernardJR2001Impaired insulin- receptor autophosphorylation is an early defect in fat-fed insulin resistant rats. American Journal of Physiology9122402247.

  • YuanMKonstantopoulosNLeeJHansenLLiZWKarinMShoelsonSM2001Reversal of obesity – and diet – induced insulin resistance with salicylates or targeted disruption of IKKβ. Science29316731677.

  • YusufSDagenaisGPogueJBoschJSleightP2000Vitamin E supplementation and cardiovascular events in high-risk patients: the Heart Outcomes Prevention Evaluation Study Investigators. New England Journal of Medicine342154160.

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    Antioxidants improve insulin sensitivity in fat-fed rats and H2O2-treated rat L6 myotubes: male Wistar rats were fed with specific diet for 9 weeks (filled square, control (n=9); filled diamond control+antioxidants (n=10); filled triangle high fat diet (HFD; n=8); X HFD+antioxidants (AO; n=8)). At the end of 9 weeks of treatment, (a) i.p. glucose tolerance test and (b) i.p. insulin tolerance test were performed as described in Materials and Methods. Data are expressed as means with their standard errors indicated by vertical bars. Area under curve (AUC) of different groups calculated from the graph using NCCS software. The AUC for the glucose tolerance test of control, control+antioxidants, HFD, and HFD+antioxidants was 15 087±894, 14 543±702, 22 333±1453*, and 16 108±769 respectively. The AUC for the insulin tolerance test of control, control+antioxidants, HFD, and HFD+antioxidants was 4352±239, 4416±227, 5816±263*, and 4442±232 respectively.*P<0.01 compared with control and antioxidant-treated groups. (c) Rat L6 myotubes were pre-treated with antioxidants for 18 h and then exposed to H2O2 for 12 h. 2-Deoxy-14C-d-glucose uptake was measured in myotubes after insulin stimulation as described in the Materials and Methods. Data represent mean±s.e.m. of three independent experiments done in triplicate. *P<0.001 in comparison with basal glucose uptake.

  • View in gallery

    Antioxidants inhibit IRS-1 serine phosphorylation and preserve insulin-stimulated IRS-1 tyrosine phosphorylation. Insulin signaling was investigated by western blotting and immunoprecipitation analysis in male Wistar rat skeletal muscle homogenates and rat L6 myotube lysates as described in Materials and Methods. (a) Immunoprecipitation and immunoblotting analysis of IRS-1 tyrosine phosphorylation in male Wistar rats. (b) Immunoprecipitation and immunoblotting analysis of IRS-1 serine phosphorylation in male Wistar rats. (c) Immunoprecipitation and immunoblotting analysis of IRS-1 tyrosine phosphorylation in rat L6 myotubes. (d) Immunoprecipitation and immunoblotting analysis of IRS-1 serine phosphorylation in rat L6 myotubes. A representative immunoblot of three independent experiments is shown. Results shown are mean±s.e.m. of three experiments. C=control, C+A=control+antioxidant, H=HFD, H+A=HFD+antioxidant, and AO=antioxidant pre-treatment. *P<0.001 compared with basal. P<0.001 compared with insulin-stimulated control and insulin-stimulated antioxidant groups. $P<0.01 compared with control and antioxidant groups.

  • View in gallery

    Antioxidants inhibit JNK pathway in fat-fed rats and H2O2-treated rat L6 myotubes. Western blotting analysis of JNK pathway was performed in (a) male Wistar rat skeletal muscle homogenates and (b) rat L6 myotube lysates as described in Materials and Methods. A representative immunoblot of three independent experiments is shown. Results shown are mean±s.e.m. of three experiments. C=control, C+A=control+antioxidant, H=HFD, H+A=HFD+antioxidant, and AO=antioxidant pre-treatment. *P<0.001 compared with control and antioxidant groups.

  • View in gallery

    Antioxidants preserve the redox balance in fat-fed rats and H2O2-treated rat L6 myotubes. (a) RT-PCR analysis of antioxidant enzymes in skeletal muscle mRNA of male Wistar rats. A representative gel picture of four independent experiments is shown. Results shown are mean±s.e.m. of four experiments. C=control, C+A=control+antioxidant, H=HFD, H+A=HFD+antioxidant. *P<0.001 compared with control and antioxidant groups. (b) Total antioxidant status in cell lysates of rat L6 myotubes. (c) Reduced glutathione levels in cell lysates of rat L6 myotubes. AO = antioxidant pre-treatment. Data represent mean±s.e.m. of three independent experiments done in triplicate.*P<0.01 compared with control and antioxidant-treated cells. GPX, glutathione peroxidase; GSH, glutathione; TEAC, Trolox equivalent antioxidant capacity.

  • View in gallery

    Proposed mechanism for insulin resistance and beneficial effect of antioxidants in high-fat-fed rats.

References

AdlerVYinZTewKDRonaiZ1999Role of redox potential and reactive oxygen species in stress signaling. Oncogene1861046111.

AebiH1984Catalase in vitro. Methods in Enzymology105121126.

AgirreVUchiudaTYenushLDavisRWhiteMF2000The c-Jun NH(2) terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser (307). Journal of Biological Chemistry2773001030018.

AnnaDAnitaOFredrikHNPeterS2005Attenuation of insulin – stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes. Journal of Biological Chemistry2803438934392.

BakkerSJIjzermenRGTeerlinkTWesterhoffHVGansROHeineRJ2000Cytosolic triglycerides and oxidative stress in central obesity: the missing link between excessive atherosclerosis, endothelial dysfunction, and β-cell failure?Atherosclerosis1481721.

BeutleEDuronOKellyBM1963Improved method for the determination of blood glutathione. Journal of Laboratory and Clinical Medicine61882888.

BhorVMRaghuramNSivakamiS2004Oxidative damage and altered antioxidant enzyme activities in the small intestine of streptozotocin-induced diabetic rats. International Journal of Biochemistry and Cell Biology368997.

BlairASHajduchELitherlandGJHundalSH1999Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress. Journal of Biological Chemistry2743629336299.

BoverisA1984Determination of the production of superoxide radicals and hydrogen peroxide in mitochodria. Methods in Enzymology105429435.

BradfordM1976A rapid and sensitive method for the quantification of microgram quantity of protein utilizing the principle of protein dye binding. Analytical Biochemistry72248254.

BuettnerRScholmerichBBollheimerC2007High-fat diets: modeling the metabolic disorders of human obesity in rodents. Obesity15798808.

CarissonGBorgLAWelshN1999Sodium palmitate induces partial mitochondrial uncoupling and reactive oxygen species in rats pancreatic islets in vitro. Endocrinology14034223428.

ChanceBSiesHBoverisA1979Hydrogen peroxide metabolism in mammalian organs. Physiological Reviews59527605.

DrabkinDLAustinJN1932Spectrophotometric constants for common hemoglobin derivatives in human, dog and rabbit. Journal of Biological Chemistry98719733.

EvansJLGoldfineID2000α-Lipoic acid: a multi-functional antioxidant that improves insulin sensitivity in patients with type 2 diabetes. Diabetes Technology and Therapeutics2401413.

EvansJLGoldfineIDMadduxBAGrodskyGM2002Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocrine Reviews23599622.

FerraiMFornasieroMCIsettaAM1999MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. Journal of Immunological Methods131165172.

HansenPAHanHDMarshallBANolteLAChenMMMuecklerMHolloszyOJ1998A high fat diet impairs stimulation of glucose transport in muscle. Functional evaluation of potential mechanisms. Journal of Biological Chemistry2732615726163.

HirosumiJTunemanGChangLGorgunCZUysalKTMaedaKKarinMHotamisligilGS2002A central role for JNK in obesity and insulin resistance. Nature420333336.

JacobSLehmannRRettKHaringHU2000Oxidative stress and insulin action: a role for antioxidant. In Antioxidant in Diabetes Management pp 319338. Eds PackerLRosenPTritschlerHKingGL. New York: Marcel dekker.

KanetoH2005The JNK pathway as a therapeutic target for diabetes. Expert Opinion on Therapeutic Targets9581592.

KrssakMPetersenKFDresnerA1999Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: H-1 NMR spectroscopy study. Diabetes42113116.

LaemmliUK1970Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature227680685.

LengYKarlssonHKZierathJR2004Insulin signaling defects in type 2 diabetes. Reviews in Endocrine and Metabolic Disorders5111117.

LiuGRondinoneCM2005JNK bridging the insulin signaling and inflammatory pathway. Current Opinion in Investigational Drugs6979987.

MadduxBASeeWLawrenceJCJrGoldfineALGoldfineIDEvansJL2001Production against oxidative stress induced insulin resistance in rat L6 muscle cells by micromolar concentrations of α-lipoic acid. Diabetes50404410.

MaxwellSRJThomansonHSandlerDLeguenCBaxterMAThorpeGHJonesAFBarnettAH1997Antioxidant status in patients with uncomplicated insulin-dependent and non -insulin dependent diabetes mellitus. European Jouranl of Clinical Investigation27487490.

MotheIObberghenVE1996Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action. Journal of Biological Chemistry2711122211227.

NakataniYKanetoHKawamoriDHatazakiMMiyatsukaTMatsuokaTAKajimotoYMatsuhisaMYamasakiYHoriM2004Modulation of the JNK pathway in liver affects insulin resistance status. Journal of Biological Chemistry2794580345809.

OparaECAbdel RahmanESolimanEKamelWASoukaSLoweJEAbdel-AleemS1999Depletion of total antioxidant capacity in type 2 diabetes. Metabolism4814141417.

OtczalkLSchonfeldP1993Effect of fatty acids on energy coupling processes in mitochondria. Biochemica et Biophysica Acta11834157.

PanDALilliojaSKriketosADMilnerMRBaurLRBogardusCJenkinsABStorlienLH1997Skeletal muscle triglyceride levels are inversely related to insulin action. Diabetes46983988.

PazKHemiRLeRoithDKarasikAElhananyEKanetyHZiokY1997A molecular basis of insulin resistance: elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the jextamembrane region of the insulin receptor and impairs their ability to undergo insulin induced tyrosine phosphorylation. Journal of Biological Chemistry2722991129918.

PerseghinGGhoshSGerowKShulmanGI1997Metabolic defects in lean nondiabetic offspring of NIDDM parents: a cross-sectional study. Diabetes4610011009.

PhillipsDICaddySIIicVFieldingBAFraynKNBorthwickACTaylorR1996Intramuscular triglycerides and muscle insulin sensitivity: evidence for a relationship in nondiabetic subjects. Metabolism45947950.

PickavanceLCWildingJP2007Effects of S 15511, a therapeutic metabolite of insulin sensitizing agent S15261, in Zucker Diabetic Fatty rat. Diabetes Obesity and Metabolism9114120.

PiroSAnelloMDi PietroCLizzioMNPatane`GRabuazzoAMVigneriRPurelloMPurelloF2002Chronic exposure to fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. Metabolism5113401347.

PurvesTMiddlemasAAgthongSJudeEBBoulronAJFeroyhoughPTomlinsonDR2001A role for mitogen-activated protein kinases in the etiology of diabetic neuropathy. FASEB Journal1525082514.

QiaoLYGoldbergJLRussellJCSunXJ1999Identification of enhanced serine kinase activity in insulin resistance. Journal of Biological Chemistry2741062510632.

RaoMSReddyJK2001Peroxisomal β-oxidation and steatohepatitis. Seminars in Liver Disease214355.

LeRoithDZickY2001Recent advances in our understanding of insulin action and insulin resistance. Diabetes Care24588597.

RudichAKozlovskyNPotashnikRBashanN1997Oxidant stress reduces insulin responsiveness in 3T3LI adipocytes. American Journal of Physiology35E935E940.

RudichATiroshAPotashnikRKhamaisiMBashanN1999Lipoic acid protects against oxidative stress induced insulin resistance impairment in insulin stimulation of proteinkinase B and glucose transport in 3T3L1 adipocytes. Diabetologia42949957.

SaadMJAArakiEMiralpeixMRothenbergPLWhiteMFKohnCR1992Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance. Journal of Clinical Investigation9018391849.

SaltielARKahnCR2001Insulin signaling and the regulation of glucose and lipid metabolism. Nature414799806.

ShigetaYHiraizumiGWadaMOjiKYoshidaT1961Study on the serum levels of thioctic acid in patients with various diseases. Journal of Vitaminology74852.

SpiegelmanBMFlierJS2001Obesity and the regulation of energy balance. Cell104531543.

StorlienLHJamesDEBurleighKMChisholmDJKraegenEW1986Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats. American Journal of Physiology. Endocrinology and Metabolism251E576E583.

SykiotisGPPapavassiouAG2001Serine phosphorylation of insulin receptor substrate -1: a novel target for the reversal of insulin resistance. Molecular Endocrinology1518641869.

TantiJFGremeauxTObberghenEMarchand-BrustelY1994Serine/threonine phosphorylation of insulin receptor substrate 1 modulates insulin receptor signaling. Journal of Biological Chemistry26960516057.

TibblesLAWoodgettJR1999The stress activated protein kinase pathways. Cellular and Molecular Life Sciences 5512301254.

VinayagamoorthiRBobbyZSelvarajNSridharMG2006Vitamin E protects the insulin sensitivity and redox balance in rat L6 muscle cells exposed to oxidative stress. Clinica Chimica Acta367132136.

WernerEDLeeJHansenLYuanMShoelsonSE2004Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302. Journal of Biological Chemistry2793529835305.

YagiK1984Assay for blood plasma or serum lipid peroxides. Methods in Enzymology1052831.

YoungrenJFJohnPBernardJR2001Impaired insulin- receptor autophosphorylation is an early defect in fat-fed insulin resistant rats. American Journal of Physiology9122402247.

YuanMKonstantopoulosNLeeJHansenLLiZWKarinMShoelsonSM2001Reversal of obesity – and diet – induced insulin resistance with salicylates or targeted disruption of IKKβ. Science29316731677.

YusufSDagenaisGPogueJBoschJSleightP2000Vitamin E supplementation and cardiovascular events in high-risk patients: the Heart Outcomes Prevention Evaluation Study Investigators. New England Journal of Medicine342154160.

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