Phenethyl isothiocyanate protects against H₂O₂-induced insulin resistance in 3T3-L1 adipocytes

Abstract

Obesity is associated with systemic oxidative stress and leads to insulin resistance. Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, has been shown to have beneficial effects in improving cellular defense activities against oxidative stress through activation of nuclear factor erythroid-2 related factor 2 (Nrf2) pathway. However, little evidence exists if the antioxidative activity has beneficial effects on glucose metabolism. Here, we tested the preventive potential of PEITC for impaired insulin-induced glucose uptake by oxidative stress in 3T3-L1 adipocytes. Treatment with PEITC increased the expression of antioxidative enzymes regulated by Nrf2 such as γ-glutamylcysteine-synthetase, heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1 and glutathione S-transferase, and reduced oxidative stress induced by H₂O₂. Furthermore, PEITC restored impaired insulin-stimulated glucose uptake, translocation of glucose transporter 4 and insulin signaling by H₂O₂. These results indicate that PEITC protected insulin-regulated glucose metabolism impaired by oxidative stress through the antioxidative activity in 3T3-L1 adipocytes.

Graphical abstract

Phenethyl isothiocyanate (PEITC) protects from insulin resistance induced by oxidative stress in 3T3-L1 adipocytes.

Key words:

• adipocytes
• insulin resistance
• oxidative stress
• phenethyl isothiocyanate

Obesity is associated with systemic oxidative stress.¹⁾ The increase in reactive oxygen species (ROS) impairs glucose uptake in adipocytes,²⁾ which occurs through translocation of glucose transporter 4 (Glut4) to the plasma membrane upon insulin stimulation. Akt activation and its downstream signaling are necessary for the stimulation of Glut4 translocation by insulin.³⁾ Oxidative stress reduces insulin-dependent Akt activation and Glut4 translocation, thereby impairing glucose uptake.^2,4) Cyanidin 3-glucoside, a natural antioxidant, has been reported to provide protection from H₂O₂-induced insulin resistance in 3T3-L1 cells,⁵⁾ and suppression of ROS production by NADPH oxidase inhibitor improves glucose tolerance in obese mice.¹⁾ Thus, increase in antioxidative potential can be a useful approach to protect insulin-regulated glucose metabolism from oxidative stress in adipocytes.

Isothiocyanates are plant bioactives found in cruciferous vegetables. Phenethyl isothiocyanate (PEITC) is an aromatic isothiocyanate and is found in watercress as its glucosinolate, gluconasturtiin.⁶⁾ PEITC and other isothiocyanates induce phase 2 and antioxidant enzymes via a nuclear factor erythroid-2-related factor (Nrf2) pathway,⁷⁾ thereby improving cellular defense activities against oxidative stress and detrimental substances. Nrf2 is a transcription factor belonging to Cap’n’collar-type basic leucine zipper proteins.⁸⁾ When exposed to oxidative/electrophilic stress, Nrf2 regulates phase 2 antioxidant enzyme genes having the antioxidant response element in the promoter region such as γ-glutamylcysteine-synthetase (γ-GCS), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1).^9–11) In fact, PEITC activates Nrf2 pathway to attenuate lipopolysaccharide (LPS)-induced inflammation and to reduce oxidative stress using ultraviolet radiation.^12,13) Therefore, isothiocyanate possibly ameliorates impaired insulin signaling and glucose metabolism by oxidative stress through the antioxidative effect.

Among the isothiocyanates derived from dietary sources, sulforaphane, which is present in broccoli, cauliflower, and kale, has been extensively studied and shown to be beneficial for obesity and glucose metabolism. Sulforaphane attenuates obesity by inhibiting adipogenesis in high-fat diet-fed mice¹⁴⁾ and improves high-fat and high-sucrose diet-induced glucose intolerance in mice.¹⁵⁾ Furthermore, sulforaphane inhibits adipocyte differentiation by suppressing clonal expansion via cell cycle arrest in 3T3-L1 preadipocytes and induces lipolysis through hormone-sensitive lipase activation in 3T3-L1 adipocytes.^16,17) Therefore, the suppression of lipid accumulation may contribute to the ameliorative effect on glucose metabolism by isothiocyanates. However, little evidence exists if the antioxidative activity of isothiocyanate has beneficial effects on glucose metabolism and insulin signaling. Thus, the current study was undertaken to determine the preventive potential of PEITC, one of the major dietary isothiocyanates, for impaired insulin-induced glucose uptake by oxidative stress in 3T3-L1 adipocytes.

Materials and methods

Reagents

Dulbecco’s modified Eagles’ medium (DMEM with 25 mM glucose), bovine insulin, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (St. Louis, MO, USA). Dexamethasone (DEX) was obtained from Wako Pure Chemicals (Osaka, Japan). Bovine calf serum (CS) and fetal bovine serum (FBS) were purchased from Invitrogen (Grand Island, NY, USA) and BioWest (Nuaillé, France), respectively. PEITC was obtained from LKT laboratories (St. Paul, MN, USA). Anti-NQO1, anti-lamin B, anti-Akt, anti-p-Akt1/2/3 (Ser473), and anti-insulin receptor β (C-19) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-Nrf2 (D1Z9C), anti-Glut4 (1F8), anti-pAkt (Thr308, #9275) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-HO-1 antibody was obtained from Enzo Life Sciences (Plymouth Meeting, PA, USA). Anti-rat glutathione S-transferase (GST) Ya antibody (GS09) was obtained from Oxford Biomedical Research (Oxford, MI, USA). Anti-γ-GCS(GCLC, ab190685) antibody was obtained from Abcam plc (Cambridge, UK). Anti-phospho insulin receptor (Y1162/Y1163) antibody was obtained from R&D systems (Minneapolis, MN, USA). Anti-β actin antibody (AC-15) was obtained from Novus Biologicals (Littleton, CO, USA). All other reagents were of analytical grade.

Cell culture and differentiation

Mouse 3T3-L1 preadipocytes (Human Science Research Resources Bank, Osaka, Japan) were cultured in DMEM containing 10% (v/v) bovine CS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) in a humidified atmosphere of 5% CO₂ at 37 °C until confluent and then maintained in the same medium for an additional 2 days. Differentiation was induced at 2 days post-confluence by incubation in DMEM containing 10 μg/mL human recombinant insulin, 250 nM DEX, 0.5 mM IBMX, and 10% FBS for 3–4 days. The medium was replaced with DMEM containing 5 μg/mL insulin and 10% FBS and changed every 2 days. After 4 days, the cells were treated with or without 15 μM PEITC in DMEM containing 5 μg/mL insulin and 10% FBS for 48 h.

Oil Red O staining for lipids assessment

After incubation in DMEM containing 10 μg/mL insulin, 250 nM DEX, 0.5 mM IBMX, and 10% FBS for 3–4 days, the medium was replaced with DMEM containing 5 μg/mL insulin and 10% FBS with PEITC (0–20 μM) and was changed every 2 days. After 5 days of treatment with various PEITC concentrations, the same volume of 10% neutral formalin as the medium was added to the culture and the cells were mildly fixed for 20 min at room temperature. The formalin and medium were replaced with 10% neutral formalin. After another 1 h fixation, the cells were washed twice with PBS and stained with Oil Red O solution for 1 h at room temperature. The Oil Red O solution was prepared by mixing a stock solution (0.15 g of Oil Red O in 30 mL of isopropanol) with 20 mL of water, followed by filtration. After staining, the cells were washed twice with water and dried in air at room temperature. Oil Red O dye was eluted with isopropanol, and the extracted dye was quantified by measuring the absorbance at 490 nm to analyze the level of lipid content.

Determination of ROS production

The level of intracellular ROS was assessed by measuring the oxidation of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) according to a previously described method with some modifications.¹⁸⁾ DCFH-DA diffuses through the cell membrane and is enzymatically hydrolyzed by intracellular esterase to non-fluorescent DCFH. Intracellular ROS oxidize DCFH to fluorescent 2′,7′-dichlorofluorescein (DCF), whose intensity of fluorescence is directly proportional to the levels of intracellular ROS. Briefly, the cells were labeled with 10 μM of DCFH-DA (Sigma) in DMEM containing 0.1% BSA in a 24-well culture plate (5 × 10⁴ cells/well, Assist, Tokyo, Japan) for 45 min, followed by pretreatment with 15 μM of PEITC for 48 h. The cells were then exposed to H₂O₂ (0, 200 μM) in DMEM containing 0.1% BSA for 30 min. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using an Infinite F200PRO plate reader (TECAN, Grödig, Austria).

Measurement of glucose uptake

We evaluated glucose uptake from the rate of glucose consumption in medium according to previously described method with some modifications.¹⁹⁾ The differentiated 3T3-L1 cells (5 × 10⁴ cells/well in a 24-well plate) were cultured with 15 μM PEITC in DMEM containing 10% FBS and insulin. After 48 h, the cells were washed once with PBS and incubated with H₂O₂ (0–200 μM) in DMEM containing 0.1% BSA for 24 h. The cells were then incubated in phenol red and glucose-free DMEM (Sigma) containing 25 mM glucose with or without 100 nM insulin for 3 h. Glucose concentrations in the medium were determined using a glucose assay kit (Glucose CII-Test, Wako, Osaka, Japan), and the amounts of glucose uptake were calculated from the differences in glucose concentrations between before and after incubation.

Analysis of insulin signaling and western blot analysis

After 48 h treatment with 15 μM PEITC, the cells (2.5 × 10⁵ cells/35 mm dish) were washed once with PBS and incubated with H₂O₂ (0, 200 μM) in DMEM containing 0.1% BSA for 24 h. The medium was replaced with fresh DMEM containing 0.1% BSA, and the cells were stimulated with 100 nM insulin for 10 min. The medium was removed and the cells were lysed in lysis buffer solution (lysis buffer A: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% (w/v) SDS, 1% (v/v) Triton-X, 1% (w/v) deoxycholic acid, 0.5 mM Na₃VO₄, and 5 mM EDTA). Cell extracts were prepared by centrifugation for 10 min at 20,000 × g at 4 °C to remove the cell debris. Protein concentrations were measured as described previously.²⁰⁾ Equal amounts of proteins were separated on SDS-PAGE gels and then transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was immersed in blocking buffer (5% BSA in 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 1 mM EDTA, 0.1% Tween 20 [TBS-T]) at room temperature for 1 h, and then incubated overnight at 4 °C with primary antibodies, followed by incubation with the appropriate secondary antibody, i.e., anti-rabbit, anti-goat, or anti-mouse IgG antibody conjugated with horseradish peroxidase at room temperature for 1 h. Immunoreactive signals were detected using ECL Western Blotting Substrate (GE Healthcare, Tokyo, Japan) and a luminoimage analyzer (ImageQuant LAS 4000, GE Healthcare), and then analyzed with a software (NIH Image).

Measurement of nuclear translocation of Nrf2

To assess the translocation of Nrf2 into the nucleus upon PEITC treatment, the amount of Nrf2 in the nuclear fraction was determined by Western blotting with anti-Nrf2 antibody as described previously.²¹⁾ After 48 h treatment with PEITC, the cells were harvested with a 10 mM HEPES-NaOH (pH 7.4) buffer containing 10 mM KCl, 2 mM MgCl₂, 1 mM EDTA, and 0.5% NP-40. The homogenates were centrifuged at 20,000 × g for 10 min at 4 °C. The precipitate was resuspended in the lysis buffer A and centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was collected and used as the nuclear fraction.

Measurement of Glut4 translocation

To assess the translocation of Glut4 to the cell surface, the amount of Glut4 in the plasma membrane fraction was determined by Western blotting with anti-Glut4 antibody, according to the method previously described with some modifications.²²⁾ Briefly, 3T3-L1 cells were incubated with or without 100 nm insulin for 15 min and then harvested with a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM Na₃VO₄, 5 mM EDTA, and 0.1% NP-40). The homogenates were centrifuged at 20,000 × g for 10 min at 4 °C. The precipitate was lysed using a lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM Na₃VO₄, 5 mM EDTA, and 1.0% NP-40) and centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was collected and used as the plasma membrane fraction.

Statistical analysis

The data are expressed as the mean ± SE. For comparison of groups with comparable variances, one-way ANOVA and a Tukey’s post hoc test were carried out. The p values below 0.05 were considered significant.

Results

PEITC did not affect the level of lipid accumulation and stimulated the expression of detoxification and antioxidant proteins in 3T3-L1 cells

We first tested the effect of PEITC on lipid accumulation in 3T3-L1 adipocytes. As shown in Fig. 1, the lipid accumulation did not show any difference by treatment with PEITC until the concentration of 20 μM at the later stage of the culture, a stage after clonal expansion induced by treatment with differentiation inducer mixture of IBMX, DEX and insulin.

Fig. 1. Effect of PEITC on lipid accumulation.

Notes: 3T3-L1 adipocytes were treated with PEITC (0–20 μM) for 5 days in DMEM containing 10% FBS and insulin. The lipid content was measured as described under Materials and methods. Values are represented as the value relative to that of the absorbance at 490 nm in the control cells. Values are the mean ± SE (n = 4).

It has been reported that isothiocyanates induce the expression of detoxification and antioxidant genes including γ-GCS, NQO1, HO-1, and GST through Nrf2 activation.^23,24) We examined the effect of PEITC on the translocation of Nrf2 into the nucleus and on the induction of antioxidative enzymes regulated by Nrf2 in 3T3-L1 adipocytes. As shown in Fig. 2, the amount of Nrf2 in the nuclear fraction was increased in a dose-dependent manner of PEITC treatment. Concomitantly, the amount of γ-GCS, HO-1, NQO1, and GST proteins was significantly increased at the concentration of 15 μM and more of PEITC (Fig. 3). Isothiocyanates including PEITC are reported to have a cytotoxic effect in cancer cells.^25,26) Thus, we treated with 15 μM of PEITC in further studies, although no obvious changes were observed in 3T3-L1 cells treated even with 20 μM of PEITC.

Fig. 2. Effect of PEITC on Nrf2 nuclear translocation.

Notes: The cells were treated with PEITC (0–20 μM) for 48 h in DMEM containing 10% FBS and insulin. The nuclear fraction for Western blotting was prepared as described under Materials and methods. Equal amounts of protein were subjected to SDS-PAGE, and immunoblotting was performed with anti-Nrf2 and anti-lamin B antibodies. Lamin B was detected as loading control of the nuclear fraction. Values are the mean ± SE (n = 3). There are significant differences between values with different letters (p < 0.05).

Fig. 3. Effect of PEITC on the protein level of Nrf2-regulating genes.

Notes: The cells were treated with PEITC (0–20 μM) for 48 h in DMEM containing 10% FBS and insulin. Cell lysates were prepared as described under Materials and methods. Equal amounts of protein were analyzed by Western blotting with anti-γ-GCS (B), anti-HO-1 (C), anti-NQO1 (D), and anti-GST (E) antibodies. β actin was detected as a reference control for equal protein loading. Representative results are shown in (A). Values are the mean ± SE (n = 3). There are significant differences between values with different letters (p < 0.05).

PEITC reduced oxidative stress by H₂O₂ treatment

To investigate the protective effect of PEITC against oxidative stress, we next assessed the level of DCFH-DA oxidation upon treatment with H₂O₂ in 3T3-L1 cells pretreated with PEITC. As shown in Fig. 4, pretreatment with 15 μM PEITC clearly reduced the oxidative stress induced by H₂O₂ in 3T3-L1 cells.

Fig. 4. Effect of PEITC on oxidative stress in H₂O₂-exposed 3T3-L1 cells.

Notes: The intracellular oxidative stress was measured with DCFH-DA as described under Materials and methods. Values are the mean ± SE (n = 3). There are significant differences between values with different letters (p < 0.05).

PEITC protected against downregulation of glucose uptake and insulin-stimulated translocation of Glut4 in H₂O₂-exposed 3T3-L1 adipocytes

Insulin increases cell surface Glut4, and thereby, induces glucose uptake in 3T3-L1 adipocytes. Thus, we next examined the protective effect of PEITC on glucose uptake and the translocation of Glut4 to the plasma membrane by insulin in H₂O₂-exposed cells. As shown in Fig. 5(A), treatment with H₂O₂ decreased insulin-induced glucose uptake, which was restored by pretreatment with PEITC. Consistent with the glucose uptake, insulin-induced translocation of Glut4 was decreased with H₂O₂ and the impairment of insulin-induced Glut4 translocation by H₂O₂ was recovered by pretreatment with PEITC (Fig. 6(B)). Further, we analyzed whether the treatment with PEITC and H₂O₂ affects the total cellular protein level of Glut4. As shown in Fig. 6(A), pretreatment with PEITC slightly increased the total cellular amount of Glut4 protein, while that with H₂O₂ did not affect it.

Fig. 5. Effect of PEITC on insulin-dependent glucose uptake.

Notes: The cells were treated with PEITC (15 μM) for 48 h in DMEM containing 10% FBS and insulin. (A) After PEITC treatment, the cells were serum-starved for 24 h with H₂O₂ (0, 200 μM). The medium was then changed to a fresh medium for glucose uptake assay, and the cells were incubated with 100 nM insulin for 3 h as described under Materials and methods. (B) After PEITC treatment, the cells were subjected to glucose uptake assay with 100 nM insulin for 3 h without the step of H₂O₂ treatment. Values are the mean ± SE (n = 3). There are significant differences between values with different letters (p < 0.05).

Fig. 6. Effect of PEITC on the amount of Glut4 protein and insulin-dependent translocation of Glut4 protein to plasma membrane.

Notes: The cells were treated with PEITC (15 μM) in DMEM containing 10% FBS and insulin. After 48 h, the cells were serum-starved for 24 h with H₂O₂ (0, 200 μM). (A) After H₂O₂ treatment, the cells were lysed and the whole cell lysates were prepared. The amount of Glut4 protein in the cell lysates was analyzed by Western blotting. β actin was detected as a reference control for equal protein loading. (B) After H₂O₂ treatment, the medium was then changed to a fresh medium containing 100 nM insulin, and thereafter, cells were incubated for 15 min. Glut4 proteins in the plasma membrane were detected by Western blotting as described in Materials and methods. Immunoblotting with anti-insulin receptor β subunit (IR) was performed as preparation control for membrane fraction. (C) After PEITC treatment, the cells were stimulated with 100 nM insulin for 15 min without the step of H₂O₂ treatment. Glut4 proteins in the plasma membrane were detected by Western blotting. Values are the mean ± SE (n = 3). There are significant differences between values with different letters (p < 0.05).

To investigate the possibility that the increased level of Glut4 protein by PEITC contributes to restoration from the impairment of insulin-induced glucose uptake and translocation of Glut4 by H₂O₂, we next examined the effect of PEITC on glucose uptake and Glut4 translocation in 3T3-L1 cells without H₂O₂ treatment. The differentiated cells were incubated with 15 μM PEITC for 48 h, and then the medium was replaced with a fresh DMEM + 0.1% BSA. The cells were stimulated with 100 nM insulin for 3 h for glucose uptake assay and for 15 min in order to analyze translocation of Glut4. As shown in Figs. 5(B) and 6(C), PEITC alone did not have any effect on glucose uptake and the amount of Glut4 in the membrane fraction. Furthermore, any synergic effect of PEITC on insulin-dependent glucose uptake and Glut4 translocation was not observed. These results suggest that the restoration of insulin-induced glucose uptake by PEITC in the cell with H₂O₂ treatment is not due to upregulation of the protein level of Glut4, but is likely due to recovering from the decreased rate of translocation of Glut4.

Pretreatment with PEITC restored impaired insulin signaling by H₂O₂-inducing oxidative stress

Activation of Akt is necessary for insulin-stimulated translocation of Glut4^3,27) and glucose uptake,^28,29) which is impaired by oxidative stress.²⁾ Thus, we examined changes in insulin signaling to investigate the molecular mechanism underlying the protective effect of PEITC on impaired insulin-induced glucose uptake and Glut4 translocation in H₂O₂-exposed 3T3-L1 cells. As shown in Fig. 7 (left panels), treatment with H₂O₂ impaired tyrosine phosphorylation in the insulin receptor and Akt phosphorylation in response to insulin, which was restored by pretreatment with PEITC. Then, we examined the effect of PEITC on these signaling molecules of the insulin signal under the condition without H₂O₂ treatment. As a result in Fig. 7 (right panels), PEITC alone did not induce activation of these insulin signal molecules and did not have any effect on insulin-induced activation at the level of insulin receptor. Activation of Akt induced by insulin was enhanced by pretreatment with PEITC. However, the effect on Akt activation was not observed in glucose uptake (Fig. 5(B)). These findings suggest that PEITC reduced oxidative stress through antioxidative enzymes and preserved insulin signaling from oxidative stress.

Fig. 7. Effect of PEITC on insulin-dependent signaling.

Notes: The cells were treated with PEITC (15 μM) in DMEM containing 10% FBS and insulin. After 48 h, the cells were serum-starved for 24 h with H₂O₂ (0, 200 μM). (Left panels in (A) and (B)) After H₂O₂ treatment, the medium was then changed to a fresh medium containing 100 nM insulin, and thereafter, the cells were incubated for 15 min. (Right panels in (A) and (B)) After PEITC treatment, the cells were stimulated with 100 nM insulin for 15 min without the step of H₂O₂ treatment. Cell lysates were prepared as described under Materials and methods. Equal amounts of protein were analyzed by Western blotting with anti-phosphorylated insulin receptor and anti-insulin receptor β subunit antibodies (A), anti-phospho Akt Ser-473, Thr-308 and anti-Akt antibodies (B). The graphs represent the relative band intensity levels of the ratio of phosphorylated insulin receptor to insulin receptor (pIR/IR), and pAkt (Ser) or pAkt (Thr) to total Akt (pAkt/tAkt). Values are the mean ± SE (n = 3). There are significant differences between values with different letters (p < 0.05).

Discussion

ROS are generated from the cellular metabolic reaction. Oxidative stress is increased in obese subjects and the production of ROS increases in adipose tissue of obese mice. Increased oxidative stress affects the adipose tissue function, and thereby, leads to insulin resistance and impaired glucose uptake.^1,2,30) Numerous studies have shown that PEITC, one of the dietary isothiocyanates, induces cellular defense activity through the activation of Nrf2 pathway and exerts the cancer chemopreventive effect by upregulation of protective activity against oxidative stress and reactive metabolites of carcinogens.^7,31) However, little information is available about the ameliorative effect of PEITC on the metabolic dysregulation induced by oxidative stress. The current study has examined whether PEITC has preventive activity toward impaired insulin response and glucose uptake in 3T3-L1 adipocytes treated with H₂O₂.

To examine whether PEITC treatment induces the expression of antioxidant enzymes downstream of Nrf2 pathway, we observed the translocation of Nrf2 into the nucleus and the expression of antioxidative enzymes upon treatment with PEITC. We found that the amount of Nrf2 protein increased in the nuclear fraction when the cells were treated with PEITC (Fig. 2). This result is consistent with those of previous studies using another isothiocyanate. Sulforaphane induces the accumulation of Nrf2 by inhibiting its degradation,^32,33) and sulforaphane and other isothiocyanates including PEITC increase the expression of antioxidative enzymes, while antioxidative effect of PEITC decreases in Nrf2 (–/–) macrophage cells.^7,12) In this study, PEITC induced several antioxidant enzymes downstream of Nrf2 pathway and reduced oxidative stress in 3T3-L1 adipocytes (Figs. 3 and 4). Thus antioxidative effect of PEITC in 3T3-L1 adipocytes treated with H₂O₂ in the present study would be mediated by increased expression of antioxidative enzymes downstream of Nrf2 pathway. In the present study, it is not defined whether induction of these antioxidative enzymes by PEITC is directly regulated by Nrf2 pathway in 3T3-L1 cells. Further studies are needed to clarify the precise mechanism.

Several studies support the preventive effect of the downstream enzymes of Nrf2 pathway against oxidative stress. PEITC induced enzymes related to glutathione metabolism, GST, and γ-GCS (Fig. 3). GST catalyzes the conjugation of glutathione with electrophilic substrates, and γ-GCS is a limiting enzyme in the synthesis of glutathione, thereby increasing the redox capacity against oxidative stress. Some isothiocyanates are shown to protect against oxidative stress by upregulation of γ-GCS and the increased level of glutathione.²⁴⁾ Elevated expression of NQO1 by sulforaphane is also shown to exert cytoprotective activity against cisplatin-induced oxidative stress.³⁴⁾ Furthermore, in 3T3-L1 cells, expression of NQO1 decreases lipid accumulation which is associated with oxidative stress.^1,35) Thus, NQO1 might be involved in the reduction of oxidative stress by PEITC. Several reports support that HO-1 is another potential protein for the protective effect of PEITC against oxidative stress.^36–38) Omega-3 polyunsaturated fatty acids, eicosapentaenoic acid, and docosahexaenoic acid, reduce H₂O₂-induced oxidative stress through HO-1 induction in 3T3-L1 adipocytes,³⁶⁾ and HO-1 protects human melanocytes from H₂O₂-induced oxidative stress.³⁸⁾ In addition to the antioxidant effect of HO-1, modulation of insulin sensitivity by HO-1 might be involved in the effect of PEITC. It is reported that the administration of the HO-1 inducer, cobalt protoporphyrin, reduces adiposity and improves insulin resistance in obese diabetic animals, although the physiologic role of HO-1 in adipocytes has not been established.^39,40) Taken together, PEITC would reduce oxidative stress and improve insulin resistance in adipocytes exposed to H₂O₂ by induction of the above antioxidative enzymes, although further studies are needed to clarify the precise role of these enzymes in the effect of PEITC.

Besides the mechanism through the upregulation of antioxidative enzymes in the antioxidative effect of PEITC, possible mechanisms may occur through lipid metabolism and mitochondrial biogenesis. It is reported that lipid accumulation parallels ROS production in 3T3-L1 adipocytes,¹⁾ and the chronic activation of Nrf2 by administering agonists, CDDO-Im⁴¹⁾ or oltipraz,⁴²⁾ prevents high-fat diet-induced obesity in mice. Therefore, treatment with PEITC may reduce oxidative stress through suppression of lipid accumulation. In fact, Zhang et al. demonstrated that sulforaphane increased mitochondrial biogenesis and activated glucose aerobic oxidation pathway, thereby resulting in reduction of lipid accumulation.³³⁾ However, we did not observe the reduction of lipid accumulation by treatment with PEITC during the latter stage of differentiation (Fig. 1). This result is consistent with that of a previous report that treatment of sulforaphane inhibits the accumulation of triglyceride in adipocytes during the early stage of the differentiation (a stage of clonal expansion treated with IBMX, DEX, and insulin), but does not have any effect on lipid accumulation during the late stage after clonal expansion.¹⁶⁾ Taken together, it is unlikely that the reduction of oxidative stress by PEITC in this study is relevant to the effect on lipid metabolism by PEITC.

In conclusion, we demonstrated that PEITC can induce the expression of antioxidative enzymes in 3T3-L1 adipocytes and exerts protective effect against downregulation of insulin-induced glucose uptake by H₂O₂ treatment. PEITC is found in ordinary food materials, watercress, radish, and turnip,^6,31) which may act as potential tools for the prevention and treatment of dysregulation of glucose metabolism associated with oxidative stress, although its efficacy in vivo needs be proved by further study.

Author contributions

Y.I. designed the study, M.N. and Y.I. performed the experiments, M.N., Y.I., and T.N. analyzed the data and discussed results, M.N. and Y.I. wrote the manuscript. All authors read and approved the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science [grant number 22580125], [grant number 25450161].