Resveratrol bidirectionally regulates insulin effects in skeletal muscle through alternation of intracellular redox homeostasis

Life Sciences 242 (2020) 117188

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Resveratrol bidirectionally regulates insulin effects in skeletal muscle through alternation of intracellular redox homeostasis Yingyao Quana, Shengni Huaa, Wei Lib, Meixiao Zhana, Yong Lia, Ligong Lua,

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a

Zhuhai Interventional Medical Center, Zhuhai Precision Medical Center, Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Zhuhai, Guangdong 519000, PR China b Department of General Surgery, Zhuhai Precision Medical Center, Zhuhai People's Hospital, Zhuhai Hospital Affilated with Jinan University, Zhuhai, Guangdong 519000, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Resveratrol Reactive oxygen species Glutathione Skeletal muscle Insulin sensitivity

Aims: Reactive oxygen species (ROS) bidirectionally regulate insulin sensitivity in skeletal muscle. Insulin-induced ROS generation elevates insulin-regulated metabolic effects; however, chronic oxidative stress causes severe insulin resistance in skeletal muscle. Resveratrol (RV), as a natural antioxidant, eliminates intracellular ROS. It's unclear that whether it has different roles in insulin signaling pathway in skeletal muscle. Main methods: C57BL/6J mice and C2C12 myotubes were used to assess metabolic regulation effects of RV. Protein activation was detected using Immunofluorescence and Western Blot analysis. ROS were analyzed using confocal microscope and flow cytometry sorting (FACS). Intracellular reducing molecules were detected using an enzymatic method. Glucose uptake was measured using a fluorescent deoxyglucose analog (2-NBDG). Key findings: We found that RV attenuated insulin-stimulated AKT phosphorylation via elimination of insulininduced ROS generation in skeletal muscle, suggesting that RV decreased activation of the insulin-induced AKT signaling. In skeletal muscle of insulin resistance, RV reduced oxidative stress, restored intracellular glutathione (GSH) level, and enhanced insulin-induced AKT activation and glucose absorption. These results suggested that RV ameliorated insulin resistance by change of redox levels in skeletal muscle. Significance: This study revealed bidirectional regulation effects of RV on insulin-stimulated metabolism in skeletal muscle through alternation of intracellular redox homeostasis, which might provide a guidance role for treatment of metabolic diseases.

1. Introduction Skeletal muscle is a major organ for locomotion and glucose homeostasis, comprises approximately 40% of total body mass in nonobese subjects [1]. Skeletal muscle with insulin stimulation can absorb about 80% blood glucose and increase glycogen synthesis [2]. Mechanismly, insulin receptor (IR) combined with insulin phosphorylates and activates insulin receptor substrate (IRS), subsequently activating the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB, AKT) pathway, which is responsible for most of the metabolic actions of insulin [3]. Insulin meanwhile activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the plasma membrane, leading to intracellular reactive oxygen species (ROS) generation [4]. ROS inactivate protein tyrosine phosphatases (PTPs), such as phosphatase and tensin homolog (PTEN), protein tyrosine phosphatase1B (PTP1B), which dephosphorylate tyrosyl-phosphorylated proteins and block the insulin-regulated metabolic pathway [5]. Thus, insulin⁎

induced ROS production can elevate insulin-regulated glucose metabolism. Free fatty acids (FFA) are important energetic materials during fasting, which are produced by the hydrolysis of triglycerides in the liver and adipose tissue [6,7]. Chronic FFA exposure impairs several aspects of skeletal metabolism, including glucose uptake [8], glycogen synthesis [9], lipid deposition [10], and mitochondrial function [11,12]. One of important reasons of the metabolic disorders is that excessive FFA induces ROS production and oxidative stress via mitochondrial dependent β-oxidation or microsomal enzymes [12,13]. Glucose oxidase, which catalyzes the conversion of glucose to glucuronic acid and H2O2, can cause insulin resistance in skeletal muscle [14]. However, antioxidant drugs, such as N-acetyl-cysteine (NAC) and taurine, significantly decrease oxidative stress, ameliorating insulin resistance [15,16]. These studies indicate that decrease of intracellular oxidative stress improves insulin resistance in skeletal muscle. Resveratrol (3,5,4′-trihydroxy stilbene, RV) is a natural stilbenoid,

Corresponding author. E-mail address: [email protected] (L. Lu).

https://doi.org/10.1016/j.lfs.2019.117188 Received 11 November 2019; Received in revised form 15 December 2019; Accepted 16 December 2019 Available online 19 December 2019 0024-3205/ © 2019 Elsevier Inc. All rights reserved.

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as described previously [26]. In brief, C2C12 myotubes serum-starved for 12 h and fresh tibialis anterior (TA) muscle from 6-week-old C57BL/ 6J mice were washed, then incubated in 10 mM 2-NBDG buffer (20 mM HEPES, pH 7.4, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, and 1 mM CaCl2) for 30 min at 37 °C. The cells were extracted. 2-NBDG fluorescence (Excitation: 494 nm; Emission: 551 nm) was measured by a microplate fluorimeter (Infinite M200; Tecan, Hillsborough, USA).

which has protective roles in response to injury and pathogen infection in several plants [17]. In 1939, RV was first mentioned and isolated from Veratrum album by Japanese article Michio Takaoka [18]. So far, RV has been used to studies and therapies of several diseases, including cancer [19], neurological diseases [20,21], cardiovascular disease [22], diabetes [23] and so on. RV has an intrinsic antioxidative property that could be related to its chemopreventive effects [24]. Therefore, RV can eliminate intracellular ROS and change redox homeostasis. ROS display biphasic effects in insulin-modulated glucose metabolism in skeletal muscle. Then whether dose RV have bidirectional regulation roles in skeletal muscle insulin sensitivity? In the present study, we elucidate RV effects on insulin-stimulated metabolic regulation in skeletal muscle by changing intracellular redox homeostasis, which provides further demonstration that RV controls and treats type 2 diabetes.

2.5. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) detection C2C12 myotubes were starved for 12 h in free-serum DMEM medium. The cells were treated with 10 nM insulin for 15 min or/and 50 μM RV for 30 min, and then lysed. Fresh TA was isolated from 6week-old C57BL/6J mice after overnight fast (12 h), treated with insulin and RV, then lysed. Intracellular PIP3 content was determined by PIP3 ELISA analysis (Enzyme-linked Biotechnology Co., Shanghai, China) following the supplier's instructions.

2. Materials and methods 2.1. Reagents

2.6. ROS assay RV, N-Acetylcysteine (NAC), L-buthionine-sulfoximine (BSO), Nethylmaleimide (NEM), palmitate, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) and 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) were purchased from Sigma-Aldrich (MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and horse serum were purchased from GIBCO (Grand Island, USA). Insulin was purchased from Tocris Bioscience (Ellisville, USA). 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and MitoTracker Red were purchased from Thermo Fisher Scientific (PA, USA). Anti-phospho-Ser473-AKT antibody, anti-total-AKT antibody and anti-PTEN antibody were purchased Cell Signaling Technology (Beverly, MA). Anti-β-actin antibody was obtained from Santa Cruz Biotechnology (CA, USA). Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 555-conjugated goat anti-rabbit secondary antibodies were purchased from Invitrogen (Carlsbad, USA). Goat anti-mouse IgG H&L (Alexa Fluor 680) and goat anti-rabbit IgG H&L (Alexa Fluor 790) secondary antibodies were purchased from Abcam (MA, USA).

H2DCFDA is an oxidation sensitive probe, and is cleaved by nonspecific esterases to transform into the dichlorodihydrofluorescein derivative H2DCF. H2DCF is oxidized and converts to DCF (Excitation: 488 nm; Emission: 510 nm), and is trapped inside the cell. Thus, H2DCFDA is often used to intracellular ROS detection in living cells [27]. C2C12 myotubes were starved for 12 h in free-serum DMEM medium, and then incubated with 20 μM H2DCFDA for 15 min after different treatments. Subsequently, the cells were digested and harvested, then examined using flow cytometry sorting (FACS, BD Biosciences, USA). In addition, C2C12 myotubes were incubated with H2DCFDA, then were detected using laser scanning microscope (LSM) 880 confocal microscope (Zeiss, Germany). ROS detection in TA was performed as our previous study [28]. H2O2 concentration in TA after different treatment was measured using the Amplite fluorimetric hydrogen peroxide assay kit (ATT Bioquest, CA, USA) according to the manufacturer's directions. The fluorescence (Excitation: 540 nm; Emission: 590 nm) was measured by a microplate fluorimeter.

2.2. Mice C57BL/6J mice were obtained from Southern Medical University, Guangzhou, China. All mice were housed under constant temperature and humidity environment with a 12-hour light and 12-hour dark cycle. Male mice at 4 weeks of age were fed by a high-fat diet (HFD, 60% fat). The mice fed with a normal chow diet (NCD) were the control group. HFD-induced mice for 6 weeks were intraperitoneally injected with 20 mg/kg RV (10% ethanol/PBS) or vehicle daily for 20 days. Body weight and fasting blood glucose were measured. All animal experimental protocols were performed with the approval of the Animal Use and Care Committee of Jinan University.

2.7. GSH measurement Reduced and oxidized glutathione (GSH and GSSG) were detected as our previous study [28]. C2C12 myotubes and TA after different treatments were lysed. Total GSH, GSH and GSSG contents were detected using an enzymatic method according to the commercial assay kit procedure (Beyotime Institute of Biotechnology, Jiangsu, China). 2.8. Immunofluorescence Immunofluorescence analysis was performed as described previously [29]. In brief, C2C12 cells were cultured and differentiated in 35 mm confocal dish, and treated with different treatments. The myotubes were then fixed with 4% paraformaldehyde, permeabilized by 0.5% Triton-X-100. Subsequently, the cells incubated with 5% bovine serum albumin (BSA), specific primary antibodies and fluorescence secondary antibodies in turn. For cell nucleus staining, the myotubes were incubated with 1 μg/ml DAPI for 30 min. Immunofluorescence imaging was detected using the LSM880 confocal microscope.

2.3. Cell culture C2C12 myoblasts were obtained from American Type Culture Collection (ATCC, Manassas, USA). The cells were cultured in DMEM medium containing 10% FBS, penicillin (100 Us/ml), and streptomycin (100 mg/ml) in a humid atmosphere (95% air and 5% CO2) at 37 °C. Differentiation of C2C12 myoblasts was described previously [25]. In brief, the myoblasts were cultured until 80% confluency, then maintained in a differentiation DMEM medium containing 2% horse serum, 100 U/ml penicillin, and 100 μg/ml streptomycin for 4–6 days. For FFA treatment, C2C12 myotubes were incubated with differentiation medium containing 1 mM palmitate for 24 h.

2.9. Western blot Oxidative modifications of PTEN and western blot were examined as described previously [30]. Briefly, cells were lysed using RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% NP-40, 5 mM EDTA, and 0.5% sodium deoxycholate, and 1 mM phenylmethylsulfonyl fluoride) supplemented with 10 mM NEM. The free

2.4. Glucose uptake Glucose uptake was measured using a fluorescent glucose 2-NBDG 2

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treatment (Fig. 2F). Insulin-induced PIP3 generation reduced by RV treatment was also found in TA (Fig. 2G). BSO, as an inhibitor of glutathione synthesis, enhances insulin-induced ROS generation [34], leading to increase of insulin-stimulated PIP3 production and 2-NBDG uptake as compared with single insulin-treated group (Fig. 2G, H). Next, we assessed change of reduced and oxidized GSH levels in C2C12 myotubes. Total GSH and GSH/GSSG ratio were decreased after insulin treatment, but the insulin effects were inhibited by RV and enhanced by BSO (Fig. 2I, J). These results indicated that RV suppressed change of insulin-induced redox homeostasis, attenuating insulin-induced metabolic regulation in skeletal muscle.

thiol group was alkylated by NEM, and its oxidation was blocked. Total cell lysates were subjected to non-reducing sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, MA, USA). Subsequently, the membranes were incubated with 5% BSA, the primary antibodies and the fluorescence secondary antibodies in turn. The imaging was performed using an Odyssey Infrared Imaging System (LI-COR, USA). The intensity of the western blot signals was quantitated using Image J software (NIH, Bethesda, USA). 2.10. Statistical analysis Data were presented as mean ± SEM from at least three independent experiments and analyzed using Student's t-test. Statistical analysis was performed with SPSS 19.0 software (SPSS, Chicago, USA). Significant differences were considered statistically significant at p < 0.05.

3.3. RV improves insulin sensitivity in insulin-resistant skeletal muscle Previous studies have demonstrated that chronic FFA treatment causes skeletal muscle insulin resistance [35,36]. Chronic FFA treatment significantly reduced phosphorylation of insulin-stimulated AKT in C2C12 myotubes (Fig. 3A, B). Insulin-induced 2-NBDG absorption was also lower in FFA-treated C2C12 myotubes than FFA-untreated myotubes (Fig. 3C). These results indicated that chronic FFA incubation reduced insulin sensitivity in C2C12 myotubes. However, RV treatment enhanced insulin-induced AKT phosphorylation in FFA-treated C2C12 myotubes, indicating that RV improved insulin sensitivity in FFA-induced skeletal muscles (Fig. 3D, E). We further observed that intracellular 2-NBDG level was increased in FFA-treated C2C12 myotubes with RV and insulin co-treatment as compared with insulin treatment (Fig. 3F). In male C57BL/6J mice, fasting blood glucose and body weight were significantly increased after an HFD for 6 weeks (Fig. 3G, H). Then the HFD-fed mice were treated with RV for 20 days. We found that insulin-induced 2-NBDG uptake in RV-treated HFD mice was higher than the RV-untreated group (Fig. 3I). These data indicated that RV ameliorated insulin resistance in skeletal muscle.

3. Results 3.1. RV attenuates insulin-stimulated metabolic regulation in skeletal muscle To explore RV effects in skeletal muscle, C2C12 myoblasts, which derive from the organism of a mouse and differentiate into myotubes, were experimented. Undifferentiation C2C12 myoblasts were spindleshaped or polygonal, the size uniformity (Fig. 1A). The cells elongated and fused into myotubes after differentiation of 4 days (Fig. 1A). Immunofluorescence analysis showed mononucleate C2C12 myoblasts formed multinucleate myotubes after differentiation (Fig. 1B). The data indicated that C2C12 myoblasts were differentiated into myotubes. To demonstrate whether RV affected insulin roles in skeletal muscle, we examined AKT phosphorylation in C2C12 myotubes. As shown in Fig. 1C, D, insulin treatment significantly increased AKT phosphorylation, but co-treatment with RV and insulin attenuated the effect in C2C12 myotubes. However, there was no change in glucose absorption between insulin treatment and insulin and RV co-treatment group (Fig. 1E). Because single RV treatment promoted 2-NBDG uptake, relative insulin-induced 2-NBDG uptake ability in RV-treated C2C12 myotubes (1.745 ± 0.089) was lower than the RV-untreated insulininduced group (2.228 ± 0.154) (Fig. 1E). The similar results were detected in mouse TA muscle (Fig. 1F, G). The data suggested that RV reduced insulin-induced effects in skeletal muscle.

3.4. RV changes redox homeostasis contributing to improvement of insulin resistance Previous studies revealed that elevated FFA can cause oxidative stress due to increased mitochondrial uncoupling, β-oxidation and ROS production [37,38]. Chronic oxidative stress induces insulin resistance, type 2 diabetes, and metabolic diseases [39]. Our data showed that chronic FFA treatment increased intracellular ROS levels (Fig. 4A), which contributed to reduction of insulin sensitivity in C2C12 myotubes (Fig. 3A–C). We further observed that RV significantly reduced FFA-induced ROS generation in C2C12 myotubes (Fig. 4B, C). Confocal microscope analysis also showed that RV eliminated intracellular ROS as similar as ROS scavenger NAC in C2C12 myotubes (Fig. 4D, E). Likewise, oxidative stress was attenuated in TA from RV-treated HFD mice as compared with the untreated group (Fig. 4F). Total GSH and GSH/GSSG ratio were enhanced in C2C12 myotubes (Fig. 4G, H) and TA (Fig. 4I, J) after RV treatment. To demonstrate that RV improved insulin sensitivity through decrease of intracellular ROS levels, we examined insulin-stimulated glucose uptake in C2C12 myotubes treated with RV, NAC or H2O2. The result indicated that either RV or NAC treatment promoted 2-NBDG uptake, but H2O2 treatment was no change in FFA-induced C2C12 myotubes stimulated by insulin (Fig. 4K). These results indicated that RV changed intracellular redox homeostasis, contributing to amelioration of insulin resistance in skeletal muscle.

3.2. RV reduces insulin-stimulated ROS and PIP3 generation in skeletal muscle RV has antioxidative property and scavenges intracellular ROS [31]. ROS have important roles in insulin-induced metabolic regulation [32]. We further examined intracellular redox levels in C2C12 myotubes after insulin and/or RV treatment. The FACS results indicated that insulin treatment significantly promoted intracellular ROS generation, yet RV blocked insulin-generated ROS in C2C12 myotubes (Fig. 2A, B). We also observed that RV, which inhibited insulin-induced ROS production in C2C12 myotubes using LSM880 confocal microscope, had the same effect as a ROS scavenger NAC (Fig. 2C, D). In insulin-stimulated metabolic regulation, insulin-increased intracellular ROS can oxidize and inhibit PTPs, which dephosphorylate tyrosyl-phosphorylated proteins [5]. PTEN, one member of PTPs family, dephosphorylates PIP3, which is catalytic production of PI3-K and takes part in AKT activation [33]. Thus, we detected PTEN oxidation in C2C12 myotubes with insulin and RV treatment. As shown in Fig. 2E, oxidative PTEN protein level was markedly increased after insulin treatment, but RV inhibited oxidation of insulin-induced PTEN in C2C12 myotubes. Intracellular PIP3 content was decreased in C2C12 myotubes after RV and insulin co-treatment as compared with insulin

4. Discussion ROS, as signaling regulators, have bidirectional-modulated roles in insulin signaling pathway in skeletal muscle. Insulin increases intracellular ROS generation, oxidatively inhibits PTPs activity, which elevates insulin-regulated glucose-lipid metabolism [4,5]. Mice lacking glutathione peroxidase 1 (Gpx1), which is one of the key enzymes 3

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Fig. 1. RV attenuated insulin-stimulated metabolic regulation in skeletal muscle. (A) C2C12 cell shape was detected using ordinary microscope in undifferentiated myoblasts and differentiated myotubes for 4 days. Scale bar = 100 μm. (B) Representative immunofluorescence images of C2C12 cells were detected using LSM880 confocal microscope. Green represented cytoskeleton actin; blue represented cell nucleus stained with DAPI. Scale bar = 20 μm. (C, D) Representative immunofluorescence images (C) and relative fluorescence intensity (D) of phosphorylated AKT in C2C12 myotubes. The cells were treated with 10 nM insulin for 15 min or/ and 50 μM RV for 30 min. Red represented pAKT; green represented β-actin. Scale bar = 20 μm. (E) Intracellular 2-NBDG levels in C2C12 myotubes after insulin or/ and RV (n = 3). (F, G) Phosphorylation levels of AKT (F) and 2-NBDG uptake (G) (n = 3) were detected in TA after 10 nM insulin for 15 min or/and 50 μM RV for 30 min. Data are expressed as the mean ± SEM. **p < 0.01 versus the control group; ##p < 0.01 versus the indicated group. NS, no significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. RV decreased insulin-stimulated ROS and PIP3 generation in skeletal muscle. (A, B) FACS analysis (A) and mean fluorescence intensity (MFI) quantitative analysis (B) of ROS levels in C2C12 myotubes under the indicated treatments (n = 3). (C, D) Representative DCF fluorescence images (C) and relative fluorescence intensity (D) were detected using LSM880 confocal microscope in C2C12 myotubes after different treatments. Green represented ROS stained with DCF; red represented mitochondria stained with MitoTracker Red. Scale bar = 20 μm. (E) PTEN oxidation was determined by western blot analysis in C2C12 myotubes treated with insulin or/and RV. (F) Intracellular PIP3 content was determined by PIP3 ELISA analysis in C2C12 myotubes treated with insulin or/and RV (n = 3). (G) Insulininduced PIP3 generation in TA treated with 50 μM RV for 30 min or 0.5 mM BSO for 2 h (n = 3). (H) Insulin-induced 2-NBDG uptake in TA treated with/without BSO (n = 3). (I, J) Total GSH (I) and GSH/GSSG ratio (J) in C2C12 myotubes after the indicated treatments (n = 3). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 versus the control group; #p < 0.05, ##p < 0.01 versus the indicated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. RV improved insulin sensitivity in insulin-resistant skeletal muscle. (A) Phosphorylation levels of AKT were determined by western blot analysis in C2C12 myotubes treated with 10 nM insulin for 15 min or/and 1 mM FFA for 24 h. (B) Quantitative analysis of phosphorylated AKT as described for panel Fig. 3(A) (n = 3). (C) 2-NBDG uptake in C2C12 myotubes treated with insulin or/and FFA (n = 3). (D, E) Representative immunofluorescence images (D) and relative fluorescence intensity (E) of phosphorylated AKT in FFA-induced C2C12 myotubes treated with 10 nM insulin for 15 min or/and 50 μM RV for 12 h. IR represented C2C12 myotubes of FFA-induced insulin resistance. Red represented pAKT; green represented β-actin. Scale bar = 20 μm. (F) Intracellular 2-NBDG levels in FFA-induced C2C12 myotubes treated with the indicated treatment (n = 3). (G, H) Fasting blood glucose (G) and body weight (H) in normal chow diet (NCD)/high-fat diet (HFD)fed mice for 6 weeks (n = 6). (I) Insulin-induced 2-NBDG uptake in TA from HFD-fed mice treated with or without RV for 20 days (n = 3). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 versus the control group; #p < 0.05, ##p < 0.01 versus the indicated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. RV changed redox homeostasis contributing to improvement of insulin resistance. (A) FACS analysis of intracellular ROS in C2C12 myotubes with/without FFA treatment. (B, C) FACS analysis (B) and MFI (C) of ROS levels in C2C12 myotubes treated with FFA or/and RV (n = 3). (D, E) Representative DCF fluorescence images (D) and relative fluorescence intensity (E) in C2C12 myotubes. The myotubes were treated with 50 μM RV or 100 μM NAC for 12 h. Green represented ROS stained with DCF; red represented mitochondria stained with MitoTracker Red. Scale bar = 20 μm. (F) Relative ROS levels in TA from HFD-fed mice treated with or without RV (n = 3). (G, H) Total GSH (G) and GSH/GSSG ratio (H) in FFA-induced C2C12 myotubes after RV treatment (n = 3). (I, J) Total GSH (I) and GSH/GSSG ratio (J) in TA from HFD-fed mice treated with RV for 20 days (n = 3). (K) Insulin-stimulated 2-NBDG uptake in FFA-induced C2C12 myotubes with the indicated treatment (n = 3). Data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01 versus the control group; #p < 0.05, ##p < 0.01 versus the indicated group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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resistance. These data revealed bidirectional regulation effects of RV on insulin-stimulated metabolism in skeletal muscle, which might provide a guidance role for treatment of metabolic diseases.

involved in the elimination of physiological ROS, are protected from HFD-induced insulin resistance [40]. Block of insulin-generated ROS attenuated insulin-modulated AKT activation in skeletal muscle. However, oxidative stress, or the chronic generation of ROS, causes severe insulin resistance in skeletal muscle. Chronic ROS exposure inhibits glucose uptake and decreases insulin sensitivity in C2C12 myotubes [29]. Previous studies have indicated that FFA accelerated intracellular ROS production and ER stress [37,38]. Elevated blood FFA level suppresses glucose absorption of skeletal muscle, impairing blood glucose disposal and insulin sensitivity [41]. Inhibition of oxidative stress improves glucose metabolism and insulin resistance in skeletal muscle and mouse models [42]. Pretreatment with RV promoted insulin-stimulated AKT phosphorylation and glucose uptake in insulin-resistant skeletal muscle. So RV can modulate insulin sensitivity via change of intracellular redox homeostasis in various microenvironment in skeletal muscle. A natural antioxidant RV has been reported to normalize the concentration of oxidative stress indicators such as superoxide anion, hydroxyl radical and H2O2 in diabetic rats [43,44]. Both FACS analysis and confocal microscope observed that RV reduced ROS generation in insulin-resistant skeletal muscle. Meanwhile, insulin resistance was also ameliorated in skeletal muscle after RV treatment. In diabetic rats, oral administration of RV decreases ROS levels and restores activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), which results in improvement of insulin resistance [45]. RV treatment increased GSH concentration in insulin-resistant skeletal muscle. Thus, RV improves glucose metabolism by enhancing antioxidant levels and reducing oxidative stress in skeletal muscle. Previous studies demonstrated that RV could activate NAD+-dependent deacetylases Sirtuin 1 (SIRT1) and adenosine monophosphate activated kinase (AMPK) [46,47], contributing to elevation of skeletal muscle insulin sensitivity in diet-induced insulin-resistant mice [47,48]. Activation of SIRT1 and AMPK promotes glucose uptake in skeletal muscle [49]. We found that single RV treatment promoted glucose uptake in both C2C12 myotubes and skeletal muscle. The reason led to no change of glucose uptake between co-treatment with RV and insulin and single treatment with insulin, although activation of insulin-induced AKT was attenuated in skeletal muscle after RV treatment. Although we cannot exclude effects of the RV-activated SIRT1 and AMPK signaling on amelioration of insulin resistance, this study elucidated that RV regulated insulin-stimulated AKT signaling through change of redox homeostasis in skeletal muscle. In recently years, many studies have found that RV significantly ameliorates glucose-lipid metabolism and insulin resistance in type 2 diabetes animal models and patients [50–52]. RV not only restores metabolic abnormalities and insulin sensitivity in skeletal muscle, but also ameliorates function of islet β cells [53], liver [43] and adipose tissue [54]. Improvement of these tissues and organs involved in glucose-lipid metabolism contributes to decrease of blood glucose and whole-body insulin resistance in type 2 diabetic animal models and patients. Diabetic complications are also attenuated with RV treatment, including diabetic retinopathy [55], diabetic neuropathy [56], diabetic nephropathy [57] and cerebrovascular dysfunction [58]. There results suggest that RV has a potential value to treat type 2 diabetes in the nearest future.

Author contribution YQ and LL contributed to conception and design of the research. YQ, SH, WL, MZ, and YL performed the experiments and analyzed the data. YQ and SH prepared the figures. YQ and LL interpreted the results of experiments. YQ drafted the manuscript. LL approved the final version of manuscript. Funding This work was supported by , National Key Research and Development Program of China (No. 2017YFA0205200), China Postdoctoral Science Foundation (No. 2019M653270), National Natural Science Foundation of China (No. 81571785, 81771957, 81502642), and Natural Science Foundation of Guangdong Province, China (No. 2016A030311055, 2016A030313770, 2018A030313074). Declaration of competing interest No potential conflict of interest was reported by the authors. References [1] F. Rinaldi, R.C. Perlingeiro, Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks, Transl. Res. 163 (4) (2014) 409–417. [2] H.M. O’Neill, et al., AMPK phosphorylation of ACC2 is required for skeletal muscle fatty acid oxidation and insulin sensitivity in mice, Diabetologia 57 (8) (2014) 1693–1702. [3] C.M. Taniguchi, et al., Critical nodes in signalling pathways: insights into insulin action, Nat. Rev. Mol. Cell Biol. 7 (2) (2006) 85–96. [4] J.H. Seo, et al., The major target of the endogenously generated reactive oxygen species in response to insulin stimulation is phosphatase and tensin homolog and not phosphoinositide-3 kinase (PI-3 kinase) in the PI-3 kinase/Akt pathway, Mol. Biol. Cell 16 (1) (2005) 348–357. [5] J.M. May, C. de Haen, Insulin-stimulated intracellular hydrogen peroxide production in rat epididymal fat cells, J. Biol. Chem. 254 (7) (1979) 2214–2220. [6] F. Diraison, M. Beylot, Role of human liver lipogenesis and reesterification in triglycerides secretion and in FFA reesterification, Am. J. Phys. 274 (2 Pt 1) (1998) E321–E327. [7] E.D. Rosen, B.M. Spiegelman, Adipocytes as regulators of energy balance and glucose homeostasis, Nature 444 (7121) (2006) 847–853. [8] A. Krook, et al., Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients, Diabetes 49 (2) (2000) 284–292. [9] S.E. Nikoulina, et al., Potential role of glycogen synthase kinase-3 in skeletal muscle insulin resistance of type 2 diabetes, Diabetes 49 (2) (2000) 263–271. [10] M.P. Corcoran, et al., Skeletal muscle lipid deposition and insulin resistance: effect of dietary fatty acids and exercise, Am. J. Clin. Nutr. 85 (3) (2007) 662–677. [11] D.E. Kelley, et al., Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes, Diabetes 51 (10) (2002) 2944–2950. [12] P. Schrauwen, M.K.C. Hesselink, Oxidative capacity, lipotoxicity, and mitochondrial damage in type 2 diabetes, Diabetes 53 (6) (2004) 1412–1417. [13] A.R. Martins, et al., Mechanisms underlying skeletal muscle insulin resistance induced by fatty acids: importance of the mitochondrial function, Lipids Health Dis. 11 (2012) 30. [14] P.L. Tan, et al., Differential thiol oxidation of the signaling proteins Akt, PTEN or PP2A determines whether Akt phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes derived from skeletal muscle, International Journal of Biochemistry & Cell Biology 62 (2015) 72–79. [15] C. Blouet, Meal cysteine improves postprandial glucose control in rats fed a highsucrose meal, Journal of Nutritional Biochemistry 18 (8) (2007) 519–524. [16] C.A. Haber, et al., N-acetylcysteine and taurine prevent hyperglycemia-induced insulin resistance in vivo: possible role of oxidative stress, Am. J. Physiol. Endocrinol. Metab. 285 (4) (2003) E744–E753. [17] K.P.L. Bhat, et al., Biological effects of resveratrol, Antioxid. Redox Signal. 3 (6) (2001) 1041–1064. [18] J.M. Sales, A.V. Resurreccion, Resveratrol in peanuts, Crit. Rev. Food Sci. Nutr. 54 (6) (2014) 734–770. [19] B.B. Aggarwal, et al., Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies, Anticancer Res. 24 (5A) (2004) 2783–2840. [20] Q. Wang, et al., Resveratrol protects against global cerebral ischemic injury in gerbils, Brain Res. 958 (2) (2002) 439–447. [21] Y.K. Gupta, et al., Protective effect of resveratrol against intracortical FeCl3-induced model of posttraumatic seizures in rats, Methods Find. Exp. Clin. Pharmacol.

5. Conclusion Our study focused on functions of RV in insulin-stimulated metabolic regulation of skeletal muscle through alteration of intracellular redox homeostasis. We found that RV attenuated insulin-stimulated AKT phosphorylation via elimination of insulin-induced ROS generation in skeletal muscle. However, RV reduced oxidative stress, restored intracellular glutathione (GSH) level, and enhanced insulin-induced AKT activation and glucose absorption in skeletal muscle of insulin 8

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