Resveratrol improves hepatic steatosis by inducing autophagy through the cAMP signaling pathway.

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Molecular Nutrition & Food Research

Resveratrol improves hepatic steatosis by inducing autophagy through the cAMP signaling pathway Yong Zhang*, Ming-liang Chen*, Yong Zhou, Long Yi, Yan-xiang Gao, Li Ran, Shi-hui Chen, Ting Zhang, Xi Zhou, Dan Zou, Bin Wu, Ying Wu, Hui Chang, Jun-dong Zhu, Qian-yong Zhang#, Man-tian Mi# Research Center for Nutrition and Food Safety, Institute of Military Preventive Medicine, Third Military Medical University, Chongqing 400038, P. R.China * These authors contributed equally to this work. # Co-corresponding author: Qian-yong Zhang and Man-tian Mi. Research Center for Nutrition and Food Safety, Institute of Military Preventive Medicine, Third Military Medical University, 30th Gaotanyan Main Street, Shapingba District, Chongqing 400038, P. R. China; Telephone: +86 2368772305; Fax number: +86 2368772305; E-mail: [email protected] and [email protected].

Abbreviations 3-MA, 3-methyladenine; 8-CPT, 8-cyclopentyltheophylline; AC, adenylate cyclase; ACC, cetyl coenzyme A carboxylase; AMPK, AMP-activated protein kinase; ATG, autophagy-related; BafA1, bafilomycin A1 ; cAMP, cyclic adenosine monophosphate; CC, compound C; CPT-1α,carnitine palmitoyl transferase 1 alpha; CQ, chloroquine; HFD, high fat diet; LC3, light chain 3; NAFLD, non-alcoholic fatty liver disease; PA, Palmitate; PDE, phosphodiesterases; PRKA, protein kinase A; RSV, resveratrol; SIRT1, sirtuin 1; SQSTM1, sequestosome 1; SREBP, sterol regulatory element binding protein; TG, triglyceride.

Keywords: Resveratrol; NAFLD; SIRT1; hepatocyte; treatment

Received: 08-Jan-2015; Revised: 03-Apr-2015; Accepted: 09-Apr-2015 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/mnfr.201500016. This article is protected by copyright. All rights reserved.

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Abstract Scope: Resveratrol (RSV), a natural polyphenol, has been reported to attenuate non-alcoholic fatty liver disease (NAFLD); however, its underlying mechanism is unclear. Autophagy was recently identified as a critical protective mechanism during NAFLD development. Therefore, we investigated the role of autophagy in the beneficial effects of RSV on hepatic steatosis. Methods and Results: Via Oil red O staining, triglyceride and β-hydroxybutyrate detection, we found that RSV decreased palmitate-induced lipid accumulation and stimulated fatty acid β-oxidation in hepatocytes. Based on western blot assay, confocal microscopy and transmission electron microscopy, we found that RSV induced autophagy in hepatocytes, whereas autophagy inhibition markedly abolished RSV-mediated hepatic steatosis improvement. Moreover, RSV increased cAMP levels and the levels of SIRT1 (sirtuin 1), pPRKA (phosphorylated protein kinase A) and pAMPK (phosphorylated AMP-activated protein kinase), as well as SIRT1 activity in HepG2 cells. Incubation with inhibitors of AC (adenylyl cyclase), PRKA, AMPK, SIRT1, or with AC, PRKA, AMPK, or SIRT1 siRNA abolished RSV-mediated autophagy. Similar results were obtained in mice with hepatic steatosis. Conclusions: RSV improved hepatic steatosis partially by inducing autophagy in vitro and in vivo, via the cAMP-PRKA-AMPK-SIRT1 signaling pathway, which provides new evidence regarding RSV’s effects on NAFLD treatment.

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Introduction Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease worldwide which begins with simple steatosis, then developing to steatohepatitis, at last progressing to cirrhosis or cancer [1, 2]. There is currently no satisfactory therapy for NAFLD. However, accumulating evidence indicates that a large number of polyphenols naturally presenting in fruits and vegetables exert excellent health protective effects, which may be potential candidates for the treatment of NAFLD [3-5]. Resveratrol (3, 4', 5-trihydroxystilbene, RSV) as a natural plyphenol has been found to be beneficial for many metabolic diseases including NAFLD in vivo and in vitro [6, 7]. However, the underlying mechanisms are not clear. Autophagy is crucial for development, differentiation, survival, and homeostasis, and has been shown to play important roles in the progression of many diseases including NAFLD [8]. Researchers have demonstrated that autophagy can inhibit NAFLD development by degradating the intracellular hepatocyte lipid. Increased autophagy breaks more stored lipids, thereby facilitating β-oxidation or other uses of fatty acids; for instance, when autophagy was abolished by autophagy-related gene 7 (ATG7) knockout, NAFLD was developed [9, 10]. Moreover, mice with chronic obesity or insulin resistance, which were prone to NAFLD, had notably decreased hepatic autophagy indicators [11, 12]. In addition, RSV has been shown to suppress many types of cancer cells and promote longevity through autophagy induction [13, 14]. Thus, herein, we determined the possible role of autophagy in the beneficial effects of RSV on hepatic steatosis in vitro and in vivo. Sirtuin 1 (SIRT1), an important member belonging to Sir2 family, has an essential role This article is protected by copyright. All rights reserved.

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in protecting against metabolic damages caused by high fat diet (HFD) [15-17]. Recently, SIRT1 has been shown to act as a positive regulator of basal autophagy through modulating the expression of several ATGs [18], while SIRT1-dependent autophagy activation has been found to be necessary for the benefits of RSV on aging-related diseases [19]. Park et al.[20] proved that intracellular cyclic adenosine monophosphate (cAMP) may be necessary for the health-protective effects of RSV by activating AMP-activated protein kinase (AMPK) and subsequent SIRT1 activation. We previously showed that RSV could upregulate autophagy in a SIRT1-dependent manner through activation of the cAMP-protein kinase A (PRKA)-AMPK signaling pathway in endothelial cells [14]. We therefore hypothesized that the cAMP-PRKA-AMPK-SIRT1 signaling pathway may also be involved in RSV-induced autophagy in response to hepatic steatosis. As expected, our results indicated, for the first time, that RSV improved hepatic steatosis partially by inducing autophagy in vitro and in vivo, via the cAMP-PRKA-AMPK-SIRT1 signaling pathway. These results provide new direct evidence regarding the role of autophagy in the therapeutic effects of RSV on NAFLD as well as clarifying the underlying mechanisms, which may open new avenues for finding new drugs that can be applied to NAFLD treatment.


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Materials and Methods Antibodies and reagents Green fluorescent protein - light chain 3 (LC3) plasmid was kindly provided by Dr. Tamotsu Yoshimori (Department of Cell Biology, National Institute for Basic Biology, Precursory Research for Embryonic Science and Technology, Okazaki, Japan). Dulbecco’s modified eagle medium (JP01117785) and fetal bovine serum (SH30370.03) were purchased from Hyclone Laboratories (Logan, UT, USA). Cell Counting Kit-8 (CK04) was purchased from Dojindo Laboratories (Kumamoto, Japan). Dimethyl sulfoxide (D2650), EX-527 (E7034), 3-methyladenine (3-MA, M9281), bafilomycin A1 (BafA1, B1793), chloroquine (C6628), 8-CPT(C3912), compound C (P5499), Palmitate (PA, P5585), resveratrol (RSV; R5010), Oil Red O (O0625) and antibodies against LC3 (L7543) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Forskolin (S1612) and H-89 (S1643) were purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). KH7 (3834) was purchased fromTocris Bioscience (Bristol, UK). Antibodies against adenylate cyclase (AC, sc-590), AMP-activated protein kinase αA1/2 (AMPK, sc-74461), phosphorylated AMPKαA1/2 (pAMPK, sc-33524), acetyl coenzyme A carboxylase (ACCα, sc-137104), phosphorylated ACCα (pACCα, sc-271965), protein kinase A α (PRKA, sc-903), and phosphorylated PRKAα (PRKAα, sc-12905) were obtained from Santa Cruz Biotechnology (SantaCruz, CA, USA). Antibodies against SIRT1 (2028), Beclin1 (3495), ATG5 (8540) and sequestosome 1(SQSTM1, 5114) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA) and antibodies against carnitine palmitoyl transferase 1 alpha (CPT-1α, ab176320) and sterol regulatory element binding protein 1-c (SREBP1-c, ab140483) were purchased from Abcame (Cambridge, MA). Antibodies against ACTB (TA-09) were obtained from Zhongshan Jinqiao Biotechnology (Beijing, China). LysoTracker Green (L7526) and Lipofectamine 2000 transfection reagent (11668-019) were purchased from Invitrogen (Carlsbad, CA, USA).


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Animal Experiments A diet containing RSV was prepared as describe before [21]. Briefly, 50 g powder diet was weighed and well mixed with 55 mL double-distilled H2O.Then, 15 mL of 100% ethanol containing 0.2 g of RSV (for 0. 4% in the diet) added to the chow and mixed well. The control diet was added with the same volume of 100% ethanol. For ethanol evaporation, the chow was placed in a vacuum oven at 50℃ overnight. Because RSV is sensitive to light [22], the chow was kept away from light whenever possible and stored in the dark at 4℃. 8-week-old 129/SvJ mice (Jackson Laboratory, Bar Harbor, ME, USA) were first fed with either HFD (containing 60% fat) or chow diet (containing 10% fat; Research Diets, Inc., New Brunswick, NJ, USA) for 4 weeks to induce hepatic steatosis. Thereafter, HFD-fed mice were further divided into two subgroups (n=10), which were fed with chow diet or chow diet containing RSV (0.4%) for a further 4 weeks. No traces of RSV were detected in the commercial diet, using the method reported by Juan et al.[23] Mice were weighed at 8, 12, and 16 weeks of age and food intake of mice was assessed daily. At the end of the experiment, all mice were fasted for 12 h and then anesthetized with pentobarbital sodium prior to the surgical procedures. All efforts were made to minimize suffering. Liver samples were collected for subsequent measurements. Animals were maintained under a regular 12 h light period at a controlled temperature (22 ± 2°C) and received chow and water ad libitum. Animal care and treatments were conducted according to established guidelines and protocols approved by the Animal Care and Use Committee of the Third Military Medical University (Chongqing, China) (approval SYXC-2013-00012).


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Cell culture and treatment Human hepatoma HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 10% fetal bovine serum and penicillin (100 U/mL)/streptomycin sulfate (100 µg/mL) (Invitrogen) at 37°C in a humidified atmosphere enriched with 5% CO2. All experiments were performed when the cells reached about 80–90% confluence. Hepatic steatosis was induced by treating cells with PA at the indicated times and concentrations. After successfully producing a hepatic steatosis model, cells were treated with or without a series of concentrations (20, 40 and 80 µM) of RSV for a further 24 h, or with or without RSV (40 µM) for different time intervals (6, 24 and 48 h). Hepatocyes were also treated with PA (0.2 mM) for 24 h to induce steatosis, and then exposed to 3-MA (5 mM), BAfA1 (10 nM), CQ(3 µM), EX-527 (2 µM), CC (10 µM), H-89 (10 µM), or KH7 (10 µM) for 1 h before the addition of RSV for a further 24 h. Forskolin (10 µM) and 8-CPT (10 µM) were added for 24 h, respectively. Complete culture medium was used in all experiments to avoid starvation-induced autophagy. Lipid accumulation and content measurements Hepatic lipid accumulation was detected by Oil Red O staining as described previously [24]. Briefly, HepG2 cells were cultured overnight at a density of 10,000 cells per well in a 96-well microplate (Corning Life Sciences, Acton, MA; 3650) and then exposed to the indicated treatments. The cells were then fixed in 4% paraformaldehyde for 20 min, washed three times with phosphate-buffered saline and stained with 0.5 % Oil Red O for 10 min at room temperature. Images were captured under a microscope, and then cells were dried. To This article is protected by copyright. All rights reserved.

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quantify Oil Red O content levels, isopropanol was added to each sample, after shaking at room temperature for 10 min, followed by absorbance measurement at 520 nm on a monochromator microplate reader (Safire II; Tecan Group Ltd., Männedorf, Switzerland). Livers fixed with paraformaldehyde (4%) were sectioned at 10 µm in a cryostat. Sections were stained with Oil Red O dissolved in 70% isopropyl alcohol, then images were captured under a microscope. The triglyceride of liver tissues and hepatocytes were measured by a triglyceride (TG) quantification kit (ab65336; Abcame, Cambridge, MA). Statistical analyses Quantitative data are presented as mean ± SD of three experiments. The Student t-test was used to evaluate the significant differences between the experimental values of the 2 samples being compared, and comparisons among groups were performed using one-way analysis of variance (ANOVA). SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA) was used for the analysis. A P value < 0.05 was considered statistically significant and the Tukey-Kramer post-hoc test was applied if P < 0.05. Full descriptions of additional materials and methods were given in the Supporting Information.


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Results RSV improved hepatic steatosis in vitro and in vivo PA was used to produce a cell model of hepatic steatosis as described previously [25]. At concentrations up to 0.4 mM, PA had no significant effect on hepatocyte viability (Fig.S1A). However, 0.2 mM PA significantly increased the lipid content and accumulation in HepG2 cells (Fig.1A and B) and this concentration was therefore used in subsequent experiments to achieve maximal fat over-accumulation without cytotoxicity. Thereafter, cells were pretreated with 0.2 mM PA for 24 h, then treated by various RSV concentrations (10, 20, 40, 80and 100 μM) for a further 24 h. RSV at concentrations >80 μM notably decreased the viability of HepG2 cells (Fig.S1B), and the effects of 10, 20, 40 and 80 μM RSV on PA-induced hepatic steatosis were therefore determined. As shown in Fig.1C and D, RSV reduced PA-induced cellular lipid content and accumulation in a dose-dependent manner in HepG2 cells, while RSV (10, 20, 40 and 80 μM) itself had no effect on lipid content and accumulation in HepG2 cells (data not shown). Mice were fed a HFD to induce hepatic steatosis in vivo as reported previously [26]. After 4 weeks of feeding, the model group showed increases in lipid content and accumulation in the liver compared with the control group, which were markedly attenuated by the addition of RSV (0.4%) to the diet for a further 4 weeks (Fig.1E and F). RSV also reduced the weight of HFD-fed mice without significant changes in food intake, which may also represent a benefit of RSV in hepatic steatosis (Fig.S2A and B). The plasma content of RSV detected by liquid chromatography-mass spectrometry assay was higher in mice fed RSV (0.4%) than in mice fed a standard diet (Fig.S2C). Overall, these results suggested that RSV could improve hepatic steatosis both in vitro and in vivo.


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RSV induced autophagy in hepatocytes in vitro and in vivo Expression of the autophagy indicator LC3-II was detected by western blot assay in PA-stimulated HepG2 cells [27]. RSV significantly upregulated LC3-II expression and SQSTM1 degradation compared with cells treated with PA alone in all experimental conditions, which was inhibited by 3-MA (an inhibitor of the early autophagy stages, 5 mM) pretreatment (Fig.2A-C). Furthermore, to monitor autophagic fluctuation, LC3-II levels were measured in the presence of BafA1 (an autophagosome-lysosome fusion inhibitor, 10 nM) or CQ (a late-stage autophagy inhibitor, 3 µM). As expected, BafA1 or CQ challenge resulted in further accumulation of LC3-II in PA-stimulated HepG2 cells after incubation with RSV (40 µM) for 24 h compared to cells treated with BafA1 or CQ alone, respectively (Fig.2C). These results suggested that RSV treatment promoted cellular autophagic processes in PA-treated HepG2 cells. To further confirm RSV-induced autophagy in PA-stimulated HepG2 cells. The effect of RSV on autophagosome formation was tested. As demonstrated by transmission electron microscopy, RSV increased autophagosomes in PA-treated hepatocytes, but 3-MA pretreatment significantly decreased RSV-induced formation of autophagosomes (Fig.2D). Via green fluorescent protein-LC3 puncta formation assay, we found that RSV also notably increased the number of green fluorescent protein-LC3 dots compared with cells treated with PA alone, which effect was blocked in the presence of 3-MA (Fig.2E). LysoTracker Green a fluorescent lysosomes probe was also used to indirectly determine the autophagy mediated by RSV in PA-treated hepatocytes. As indicated in Fig.2F, the green fluorescence was remarkably increased by RSV relative to PA-alone treated cells (P < 0.01), which then was This article is protected by copyright. All rights reserved.

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reduced by 3-MA treatment. As shown in Fig.2G, we found that RSV increased the expression of LC3-II and the degradation of SQSTM1 in the liver tissues of mice with hepatic steatosis induced by HFD. Additionally, RSV single-treatment could also increase the expression of LC3-II and the degradation of SQSTM1 as well as the number of autophagosomes in HepG2 cells (Fig.2A-F). In conclusion, our results demonstrated that autophagy was significantly induced by RSV in hepatic steatosis models in vitro and in vivo. Autophagy mediates RSV-induced fat oxidation and reduction of intracellular lipid content in hepatocytes In order to determine whether RSV-induced autophagy is involved in its beneficial effect on hepatic steatosis. We used different inhibitors and siRNA to block autophagy and measured their effect on hepatic steatosis improvement. As shown in Fig. 3A-C and Fig.S3, RSV-induced increase of β-hydroxybutyrate and decrease of hepatic lipid accumulation were notably attenuated in the presence of 3-MA, BafA1 or CQ, or ATG5 or Beclin1 siRNA strongly indicating the involvement of autophagy in RSV-mediated fat oxidation and reduction of lipid content in HepG2 cells. Of note, RSV-induced ACC inactivation and CPT-1α expression was not markedly affected by ATG5 or Beclin1 siRNA (Fig.3D). The results indicated that the effect of ATG5 or Beclin1 siRNA on RSV-induced β-oxidation occurred specifically through inhibition of autophagy, rather than downstream pathways affecting β-oxidation. Additionally, autophagy inhibition also had no effect on RSV stimulated expression of SREBP1-c, which is necessary for fatty acid and TG synthesis (Fig.3D) [28]. Thus, we concluded that autophagy was involved in the anti-steatosis effects of RSV in hepatocytes. This article is protected by copyright. All rights reserved.

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RSV-induced autophagy was SIRT1-dependent in hepatic steatosis models in vitro and in vivo Given the importance of SIRT1 in the regulation of autophagy and RSV bioactivity, the possible role of SIRT1 in RSV-mediated autophagy was determined in hepatocytes. RSV induced SIRT1 expression in dose- and time-dependent manners in PA-stimulated-HepG2 cells (Fig.4A-D). Furthermore, RSV significantly increased SIRT1 activity in PA-stimulated HepG2 cells and in liver tissues of mice fed with HFD (Fig.4E and F). RSV also increased the expression of SIRT1 in liver tissues of mice fed with HFD (Fig.4G and H). Moreover, HepG2 cells were pretreated with the SIRT1 inhibitor EX-527or SIRT1 siRNA to further determine the role of SIRT1 in RSV-induced autophagy. As indicated in Fig.5A-D, EX-527 or SIRT1 siRNA notably abolished RSV-induced SIRT1 activation and expression thereby inhibiting RSV-mediated increase of LC3-II and SQSTM1 degradation in PA-treated HepG2 cells. We also investigated the effect of EX-527 on RSV-induced autophagosome formation. As shown in Fig.5E-G, EX-527 markedly decreased the number of autophagosomes induced by RSV. Subsequently, EX-527 or SIRT1 siRNA attenuated RSV-induced decrease of intracellular lipid content and increase of β-oxidation in PA-stimulated HepG2 cells (Fig.S4A-C). We therefore concluded that SIRT1 was involved in RSV-induced autophagy in hepatocyte steatosis models in vitro and in vivo.


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PRKA-AMPK pathway played a key role in RSV-induced SIRT1 activation in hepatocyte steatosis models in vitro and in vivo The mechanism whereby RSV regulated SIRT1 activation in hepatocyte steatosis models was also investigated. AMPK is known to increase NAD+ content and SIRT1 phosphorylation to trigger the biological effects of SIRT1 [29]. Phosphorylated-AMPK level was increased by RSV in time- and dose-dependent manners, as demonstrated by western blot analysis (Fig.6A-D), and similar results were found in liver tissues of HFD-fed mice after RSV treatment (Fig.6E and F). Furthermore, the potent AMPK inhibitor CC (10 µM) or AMPK siRNA markedly inhibited RSV-induced AMPK activation, with subsequent decreasing of SIRT1 levels (Fig.7A-D). These results suggest that RSV activated AMPK, thereby inducing SIRT1 upregulation in hepatic steatosis models. RSV also markedly increased the level of pPRKA in PA-treated hepatocytes. RSV-induced AMPK activation was similar to that induced by the PRKA activator 8-CPT, and was notably diminished in the presence of the PRKA inhibitor H-89 or PRKA siRNA in PA-treated hepatocytes (Fig.7E-H). Furthermore, RSV also induced the level of pPRKA in liver tissues of mice with hepatic steatosis induced by HFD (Fig.7I-J). We therefore concluded that the PRKA-AMPK pathway played a key role in RSV-induced SIRT1 activation thereby upregulating autophagy in hepatocyte steatosis models in vitro and in vivo.


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RSV increased cellular cAMP levels in hepatocyte steatosis models in vitro and in vivo The AC inhibitor KH7 (10 µM) and AC siRNA were used to confirm the cAMP-dependent mechanism. Cells treated with the AC activator forskolin (10 µM) were used as a positive control. cAMP levels were unchanged in hepatocytes stimulated with PA alone but were increased by RSV, similar to the effect of forskolin treatment in PA-treated hepatocytes (Fig.8A). Furthermore, RSV-induced cAMP upregulation was markedly inhibited by KH7 or AC siRNA, thereby decreasing RSV-induced PRKA activation and autophagy in PA-stimulated HepG2 cells (Fig.8B and C). RSV also increased AC expression and cAMP levels in liver tissues of HFD-fed mice (Fig.8D and E). Interestingly, RSV-induced cAMP levels were still increased when AC activity was inhibited by KH7 or AC siRNA relative to KH7- or AC siRNA -alone treatment in PA-stimulated HepG2 cells (Fig.8A). One possibility is that RSV competitively inhibited phosphodiesterases (PDEs). In conclusion, cAMP was necessary for RSV-induced PRKA-AMPK-SIRT1 activation in hepatocyte steatosis models in vitro and in vivo.


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Discussion In the present study, we found that RSV reduced intracellular lipid content and stimulated fatty acid β-oxidation by inducing autophagy in hepatocytes both in vitro and in vivo. To the best of our knowledge, this report provided the first direct evidence to demonstrate the role of autophagy in RSV-induced protection against hepatic steatosis. Autophagy has been considered as a lipolytic mechanism through a process called macrolipophagy. Portions of lipid droplets, or even whole droplets, become trapped inside the double-membrane-bound autolipophagosome vesicles and are transported to lysosomes, where they are degraded to fatty acids to fuel β-oxidation [30]. Pharmacological inhibition, silencing or knockdown of autophagy (by targeting ATG5) in hepatocytes results in an increased hepatocyte TG level and accumulation of LDs when cultured in the presence of an exogenous or endogenous lipid stimulus, which is due to impaired lipolysis (to fuel β-oxidation) and not to increased TG synthesis [31]. Moreover, hepatic autophagy indicators are markedly decreased in the ob/ob mouse model of chronic obesity, thereby increasing lipid accumulation in the liver [12]. Age-related reductions in autophagy in the liver may contribute to hepatic lipid accumulation associated with an increased incidence of metabolic syndrome, including NAFLD, in older humans [32]. Thus, therapeutic strategies aimed at increasing autophagic function may provide a new approach to preventing NAFLD and its associated pathologies. It has been found that some drugs such as caffeine may stimulate hepatic lipid metabolism by the autophagy-lysosomal pathway [33], but little information is available regarding the role of autopahgy in the anti-hepatic steatosis effect of natural polyphenols especially RSV, which may be potential candidates for the treatment of NAFLD. This article is protected by copyright. All rights reserved.

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Although RSV has been found to trigger autophagy in cells from different organisms, extend life span in nematodes, and ameliorate the fitness of human cells undergoing metabolic stress. These beneficial effects are lost when essential autophagy modulators are genetically or pharmacologically inactivated, indicating that autophagy is required for the hepatoprotective effects of RSV [13, 34]. As far as we know, there is only one study presumed that the attenuation of lipid accumulation by RSV was probably through the alteration of autophagic pathway, however, they failed to offer any direct evidence for RSV-induced autophagy in hepatocytes [35]. In the present study, we, for the first time, directly found that autophagy played an important role in RSV-stimulated fatty acid β-oxidation independing on the downstream pathways affecting β-oxidation as well as in RSV-induced decrease of hepatic lipid content. Moreover, researchers have demonstrated that RSV can attenuate NAFLD through reducing hepatic lipogenesis via inhibiting SREBP-1c, ACC and fatty acid synthase [36-40] or by inducing fat oxidation via stimulating mitochondrial biogenesis and by upregulation of CPT-1α expression [36]. Interestingly, autophagy had no significant effect on RSV-induced inhibition of SREBP-1c which was a key regulator of hepatic lipogenesis, indicating that the autopahgy-independent pathways may also contribute to the improvement of hepatic steatosis mediated by RSV. We therefore came to the conclusion that RSV improved hepatic steatosis partially by inducing beneficial autophagy in vitro and in vivo. This provided new insights into the potential mechanisms responsible for the hepatoprotective effects of RSV, in which autophagy may play an important role. Furthermore, the mechanisms behind RSV bioactivity have been studied for several decades and several targets have been identified, including SIRT1 [41]. SIRT1 has been This article is protected by copyright. All rights reserved.

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reported to be essential for protecting against HFD-induced metabolic damage. Caloric restriction is currently the most effective treatment for NAFLD, and SIRT1 protein induction may be an important mechanism for caloric restriction-induced improvements in NAFLD [42]. SIRT1 has also been shown to act as a positive regulator of autophagy through modulating the expression of several ATGs, and SIRT1-dependent autophagy activation was recently found to be necessary for the benefits of RSV and caloric restriction against aging-related diseases [19, 43]. We recently demonstrated that RSV induced autophagy in a SIRT1-dependent manner in endothelial cells [14], while the current study showed that RSV also induced autophagy via SIRT1 activation in hepatocytes, as evidenced by the inhibition of RSV-induced autophagy in the presence of EX-527 or SIRT1 siRNA. According to previous studies, SIRT1 has been considered as a target for RSV since the first report of its effects on SIRT1 activation in yeast in 2003 [44]. However, subsequent studies showed that SIRT1 was not a direct target molecule for RSV, and demonstrated that RSV indirectly activated SIRT1 through highly promiscuous interactions with multiple unrelated targets [45], among which AMPK plays an important role. AMPK is a major sensor of energy levels that regulates energy homeostasis and metabolic stress through control of several homeostatic mechanisms, including autophagy and protein degradation [46]. It can increase NAD+ content and phosphorylation of SIRT1 to trigger the biological effects of SIRT1;however, AMPK is not the direct intracellular molecular target of RSV too [47]. Recently, Park and his colleagues found that RSV may activate SIRT1 through directly inhibiting PDE4 activity, subsequent the increase of cAMP contents, thereby activating AMPK [20]. In the present study, it was found that RSV-induced autophagy and SIRT1 activation were closely related to AMPK activation This article is protected by copyright. All rights reserved.

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in hepatocytes. Although the AMPK/SIRT1 pathway has been found to be important for the health-protective effects of RSV in many other models, the current findings provided the first evidence for the crucial role of this pathway in RSV-induced autophagy in hepatocytes, thus offering new insights into the health-protective effects of RSV against NAFLD. We also investigated the involvement of cAMP in RSV-induced autophagy in hepatocytes. cAMP is an important secondary messenger that plays important roles in the regulation of physiological activities in mammalian cells through PRKA activation [48]. Recent studies have demonstrated that AMPK functions as a kinase direct downstream of PRKA, which can phosphorylates liver kinase B1 at Ser428 thereby enhancing AMPK activation [49, 50]. It has been found that the cAMP-PRKA pathway can rapidly activate SIRT1 to promote fatty acid oxidation and energy expenditure caused by pharmacological β-adrenergic agonism or cold exposure [51]. We previously demonstrated that the cAMP signaling pathway was involved in RSV-mediated increase of SIRT1 expression in endothelial cells [14], while the current findings indicated that the cAMP-PRKA pathway may also be a major signaling pathway mediating RSV-induced SIRT1 activation in hepatocytes. Additionally, we found direct evidence regarding the role of SIRT1 in RSV-medicated autophagy. Based on our present findings and published work, we conclude that the cAMP-PRKA-AMPK pathway is involved in RSV-induced autophagy to mitigate hepatic steatosis. Finally, the various health benefits of RSV has been widely confirmed, however, its poor bioavailability due to the rapid metabolism in vivo was considered as the major concern for its further use in clinical [52]. Furthermore, the bioactivity of RSV metabolites has not been This article is protected by copyright. All rights reserved.

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exactly determined. Patel et al.[53] recently showed that the sulfate-conjugated RSV may serve as an intracellular pool for RSV generation thereby inducing autopahgy; nevertheless, whether sulfate-conjugated RSV could facilitate the effects of RSV on autophagy induction in hepatocytes requires further investigation. Additionally, AMPK has been shown to regulate autophagy through inhibition of the mammalian target of rapamycin complex and activation of UNC-51-like kinase 1 [46]. However, the possible roles of the AMPK-mammalian target of rapamycin and AMPK- UNC-51-like kinase 1 pathways in RSV upregulation of autophagy in hepatocytes need further elucidation. In conclusion, there is an urgent need for safe and pharmacologic treatments in individuals with NAFLD. However, the benefitial effect of RSV on NAFLD treatment in humans is controversial. One paper reports that RSV does not improve NAFLD in humans [54], but our recent results and other published works indicated that RSV supplementation in patients with NAFLD has a beneficial effect on inflammatory biomarkers, endoplasmic reticulum stress, liver enzymes, insulin resistance as well as glucose and lipid metabolism [55-58].Thus, the exact relationship between RSV and NAFLD treatment need to be further clarified. Until now, the majority of the previous animal studies have concentrated on the preventive effect of RSV and not the therapeutic effect [59]. The present findings provided the first direct evidence for a novel, autophagy-based mechanism responsible for the beneficial effects of RSV in hepatic steatosis treatment. Autophagy was in turn mediated at least partly through activation of the cAMP-PKA-AMPK-SIRT1 signaling pathway (Fig.9). These results thus offer new insights into the potential mechanisms responsible for hepatoprotective bioactivies of RSV and are valuable for the treatment of NAFLD through diet.


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Author Contributions Y.Z., M.-L.C, Y.Z., L.Y., Q.Y.Z, M.-T.M. were involved in the study design. Y.Z., M.-L.C, Y.-X.G, L.R., S.-H.C., T.Z., X.Z., D.Z., B.W., and Y.W conducted the experiments and the statistical analyses. Y.Z., M.-L.C, H.C. J.D.Z., Q.-Y.Z and M.-T. M. wrote the first draft of the manuscript. All authors contributed to the final versi on of the manuscript. Q.-Y.Z., and M.-T. M.had primary responsibility for the final content.

Acknowledgments This work was supported by the National Natural Science Foundation of China (grant number: 30972469; 81273059), and the Key Scientific and Technological Project of Chongqing (grant number: CSTC, 2011AB5040).

Conflict of Interest The authors declare no conflicts of interest.


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Fig. 1 RSV improved hepatic steatosis in vitro and in vivo. Cells were treated with PA at 0.1, 0.2, 0.3, 0.4 or 0.5 mM for 24 h. (A) Intracellular TG content detected using a TG-content-detection kit. (B) Cells were stained with Oil Red O and viewed under a microscope at 100× magnification. Cells were cultured with PA (0.2 mM) for 24 h, and then incubated with 10, 20, 40, or 80 µM RSV. (C) Lipid accumulation in HepG2 cells was visualized under a microscope at 100× magnification. (D) Intracellular TG content measured using a TG-content-detection kit. Mice were fed a chow diet or HFD for 4 weeks to induce hepatic steatosis and then the HFD-fed mice were divided into two subgroups, which were fed either a chow diet or a chow diet containing RSV (0.4%) for a further 4 weeks. (E) Liver tissues were stained with Oil Red O and visualized under a microscope at 20× magnification. (F) Liver TG content. Values are presented as mean ± SD (n = 3)；aP < 0.05, bP < 0.01 versus the control group; cP < 0.05, dP < 0.01 versus PA- or HFD-treated group.


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Fig. 2 RSV-induced autophagy in hepatic steatosis models in vitro and in vivo. Cells were treated with PA (0.2 mM) for 24 h, and then (A) cells were incubated with RSV 20, 40, or 80 µM for a further 24 h or (B) treated with 40 µM RSV for 6, 24, or 48 h. And cells were also treated with RSV (40 µM) alone for 24 h. LC3-Ⅱand SQSTM1 expression was detected by western blot. (C) Cells were pretreated with PA (0.2 mM) for 24 h, and then incubated with RSV (40 µM) in the presence or absence of 3-MA (an inhibitor of the early autophagy stages, 5 mM), BafA1 (an autophagosome-lysosome fusion inhibitor, 10 nM), CQ (a late-stage autophagy inhibitor, 3 µM) for a further 24 h. LC3-Ⅱand SQSTM1 expression was evaluated by western blot. (D) Following fixation, cells were visualized by transmission electron microscopy. Arrows indicate autophagosomes. (E) Cells were transfected with a plasmid expressing green fluorescent protein-LC3. After 24 h, the cells were treated with PA, 3-MA and RSV as indicated in (C). Following fixation, cells were immediately visualized by confocal microscopy and the number of green fluorescent protein-LC3 dots in each cell was counted. (F) Cells were treated with PA, 3-MA and RSV as indicated in (C). Thereafter, the cells were incubated with LysoTracker Green (a fluorescent lysosomes probe, 50 nM, 15 min, and 37°C) and visualized by confocal microscopy. The average LysoTracker Green fluorescence was expressed as the mean fluorescence intensity. (G) Mice were fed a HFD for 4 weeks to induce hepatic steatosis and then divided into two subgroups, which were fed either a chow diet or a chow diet containing RSV (0.4%) for a further 4 weeks. LC3-Ⅱand SQSTM1 expression was measured. Values are expressed as the mean ± SD (n = 3). bP < 0.01 versus the control group; cP < 0.05, dP < 0.01 versus PA- or HFD-treated group; eP < 0.01 RSV and PA-co-treated group; A.U., arbitrary units. This article is protected by copyright. All rights reserved.

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Fig. 3 RSV attenuated hepatic steatosis through the induction of autophagy. HepG2 cells were transfected with Beclin1 or ATG5 siRNA. After 24 h, cells were treated with PA (0.2 mM) for 24 h, and then incubated with RSV (40 µM) for a further 24 h. And HepG2 cells were also treated with 3-MA, BafA1 and CQ as described in Materials and methods section. (A) The expression of LC3, SQSTM1, ATG5 and Beclin1 was detected by western blot. (B) β- hydroxybutyrate levels were measured by a commercially available colorimetric kit. (C) TG content was measured using a TG-content-detection kit. (D) The expression of the indicated proteins was measured by western blot. Values are expressed as the mean ± SD (n = 3). bP < 0.01 versus the control group; cP < 0.05, dP < 0.01 versus PA-treated group. eP < 0.01 versus RSV and PA cotreated group; A.U., arbitrary units.


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Fig. 4 RSV increased SIRT1 content and activity in hepatic steatosis model in vitro and in vivo. HepG2 cells were pretreated with PA (0.2 mM) for 24 h, and then (A) cells were incubated with RSV (20, 40 or 80 µM) for a further 24 h or (C) treated with 40 µM RSV for 6, 24, or 48 h. And cells were also treated with RSV (40 µM) alone for 24 h. SIRT1 expression was detected by western blot. (B) and (D) Quantification of endogenous SIRT1. (E) Cells were treated as described in (A). SIRT1 activity was then detected using a SIRT1 activity assay kit. Mice were fed a HFD for 4 weeks to induce hepatic steatosis and then divided into two subgroups, which were fed either a chow diet or a chow diet containing RSV (0.4%) for a further 4 weeks. (F) SIRT1 activity was detected using a SIRT1 activity assay kit. (G) SIRT1 expression was measured by western blot. (H) Quantification of SIRT1. Values are expressed as mean ± SD (n = 3). bP < 0.01 versus the control group; cP < 0.05, dP < 0.01 versus PA- or HFD-treated group; A.U., arbitrary units.


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Fig. 5 RSV-induced autophagy in hepatocytes was SIRT1-dependent. HepG2 cells were pretreated with PA (0.2 mM) for 24 h, and then incubated with RSV (40 µM) in the presence or absence of EX-527 (an inhibitor of SIRT1, 2 µM) for a further 24 h. (A) SIRT1 activity was detected using a SIRT1 activity assay kit. (B) SIRT1, LC3 and SQSTM1 expression was detected by western blot. HepG2 cells were transfected with SIRT1 siRNA. After 24 h, cells were treated with PA (0.2 mM) for 24 h and then incubated with RSV (40 µM) for a further 24 h. (C) SIRT1 activity was detected using a SIRT1 activity assay kit. (D) Quantification of the indicated proteins. (E) HepG2 cells were transfected with a plasmid expressing green fluorescent protein-LC3 for 24 h and then treated as described in (A). Following fixation, the cells were immediately visualized by confocal microscopy. The number of Green fluorescent protein-LC3 dots in each cell was counted. Cells were treated as described in (A). (F) Lysosomes were loaded with LysoTracker Green and visualized by confocal microscopy. The average LysoTracker Green fluorescence was expressed as the mean fluorescence intensity. (G) Cells were examined using transmission electron microscopy. Arrows indicate autophagosomes. Values are expressed as mean ± SD (n = 3). bP < 0.01 versus the control group; cP < 0.05, dP < 0.01 versus PA-treated group; eP < 0.01 versus PA and RSV co-treated group; A.U., arbitrary units.


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Fig. 6 RSV activated AMPK in hepatic steatosis models in vitro and in vivo. HepG2 cells were pretreated with PA (0.2 mM) for 24 h, and then (A) cells were incubated with RSV (20, 40 or 80 µM) for a further 24 h or (C) treated with 40 µM RSV for 6, 24, or 48 h. And cells were also treated with RSV (40µM) alone for 24 h. The level of pAMPK was detected by western blot. (B) and (D) Quantification of endogenous pAMPK. (E) Mice were fed a HFD for 4 weeks to induce hepatic steatosis and then further divided into two subgroups, which were fed either a chow diet or a chow diet containing RSV (0.4%) for a further 4 weeks. pAMPK level was measured by western blot. (F) Quantification of endogenous pAMPK. Values are expressed as mean ± SD (n = 3). bP < 0.01 versus the control group; cP < 0.05, dP < 0.01 versus PA- or HFD-treated group; A.U., arbitrary units.


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Fig. 7 RSV activated SIRT1 via the PRKA-AMPK pathway in hepatocyte steatosis models in vitro and in vivo. (A) Cells were preincubated with PA (0.2 mM) for 24 h, and then incubated with RSV (40 µM ) in the presence or absence of CC (a potent AMPK inhibitor, 10 µM) for 24 h. SIRT1 and pAMPK level was measured by western blot. (B) Quantification of the indicated proteins. (C) AMPK and (E) PRKA were knocked down by siRNA transfection. At 24 h post-transfection, the cells were treated with PA (0.2 mM) for 24 h, washed, and incubated with RSV (40 µM ) for a further 24 h. The proteins were subjected to western blot analysis. (D) and (F) Quantification of the indicated proteins. (G) Cells were preincubated with PA (0.2 mM) for 24 h, then, incubated with RSV (40 µM) or 8-CPT (a PRKA activator, 10 µM) in the presence or absence of H89 (a PRKA inhibitor, 10 µM) for 24 h. The expression of the indicated proteins was measured by western blot. (H) Quantification of the indicated proteins. Mice were fed a HFD for 4 weeks to induce hepatic steatosis and then further divided into two subgroups, which were fed either a chow diet or a chow diet containing RSV (0.4%) for a further 4 weeks. (I) pPRKA level was measured by western blot assay. (J) Quantification of pPRKA and PRKA. Values are expressed as mean ± SD (n = 3). d

P < 0.01 versus PA- or HFD-treated group; eP < 0.01 versus PA and RSV co-treated group;

A.U., arbitrary units.


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Fig. 8 RSV increased cellular cAMP levels in hepatocyte steatosis models in vitro and in vivo. (A) Cells were stimulated with PA (0.2 mM) for 24 h, and then incubated with RSV (40 µM) in the presence or absence of KH7 (an AC inhibitor, 10 µM) or AC siRNA transfection for a further 24 h. Forskolin (an AC activator, 10 µM) was added 24 h after PA treatment for a further 24 h. And cells were also treated with RSV(40 µM) alone for 24 h. The cells were then lysed and 100 µL aliquots of the cleared lysate were used for cAMP assay. (B) and (C) The expression of indicated proteins was detected by western blot. Mice were fed a HFD for 4 weeks to induce hepatic steatosis and then further divided into two subgroups, which were fed either a chow diet or a chow diet containing RSV (0.4%) for a further 4 weeks. Liver tissues were collected and lysed. (D) Liver tissue proteins were subjected to western blot analysis. (E) Aliquots of the cleared lysate (100 µL) were used for cAMP assay. Values are expressed as mean ± SD (n = 3). bP < 0.01 versus the control group; dP < 0.01 versus PA- or HFD-treated group; eP < 0.01 versus RSV and PA co-treated cells; *P < 0.05 versus KH7 and PA co-treated cells; #P < 0.01 versus AC siRNA and PA co-treated cells; A.U., arbitrary units.


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Fig. 9 Regulation of RSV-induced autophagy in hepatocytes. RSV improved hepatic steatosis partially by inducing autophagy through the proposed signaling pathways. RSV increases cAMP levels, possibly through activating AC or inhibiting the activity of PDE4, thereby activating cAMP signaling via PRKA and increasing AMPK and SIRT1 activity, and ultimately inducing autophagy.


Dihydromyricetin improves skeletal muscle insulin resistance by inducing autophagy via the AMPK signaling pathway.

choline-deficient diet-induced steatohepatitis through regulating autophagy.

Activation of autophagy attenuates EtOH-LPS-induced hepatic steatosis and injury through MD2 associated TLR4 signaling.

Activation of free fatty acid receptor 1 improves hepatic steatosis through a p38-dependent pathway.

Dihydromyricetin improves skeletal muscle insulin sensitivity by inducing autophagy via the AMPK-PGC-1α-Sirt3 signaling pathway.

Olfactory receptor 10J5 responding to α-cedrene regulates hepatic steatosis via the cAMP-PKA pathway.

Metformin ameliorates hepatic steatosis and improves the induction of autophagy in HFD‑induced obese mice.

Autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway.

Induction of autophagy and apoptosis by miR-148a through the sonic hedgehog signaling pathway in hepatic stellate cells.

Glucagon regulates hepatic lipid metabolism via cAMP and Insig-2 signaling: implication for the pathogenesis of hypertriglyceridemia and hepatic steatosis.

Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats.

Ezetimibe improves hepatic steatosis in relation to autophagy in obese and diabetic rats.

Miltefosine Suppresses Hepatic Steatosis by Activating AMPK Signal Pathway.

Resveratrol improves hepatic insulin signaling and reduces the inflammatory response in streptozotocin-induced diabetes.

Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition.

SREBP1 signaling pathway.

mTOR pathway in melanoma B16 cells.

Hepatic mitochondrial and ER stress induced by defective PPARα signaling in the pathogenesis of hepatic steatosis.

Resveratrol attenuates hepatic steatosis in high-fat fed mice by decreasing lipogenesis and inflammation.

SREBP Pathway: A Mediator of Hepatic Steatosis.

mTOR pathway.

Modified SJH alleviates FFAs-induced hepatic steatosis through leptin signaling pathways.

Combination of honokiol and magnolol inhibits hepatic steatosis through AMPK-SREBP-1 c pathway.

Signaling through NOD-2 and TLR-4 Bolsters the T cell Priming Capability of Dendritic cells by Inducing Autophagy.