Formononetin alleviates hepatic steatosis by facilitating TFEB-mediated lysosome biogenesis and lipophagy

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ScienceDirect Journal of Nutritional Biochemistry 73 (2019) 108214

Formononetin alleviates hepatic steatosis by facilitating TFEB-mediated lysosome biogenesis and lipophagy Yan Wang a, b, c , Hailing Zhao a , Xin Li a , Qian Wang a, d , Meihua Yan a , Haojun Zhang a , Tingting Zhao a , Nannan Zhang a, b , Pan Zhang a, d , Liang Peng a, b,⁎, Ping Li a, b,⁎⁎ b

a Beijing Key Laboratory for Immune-Mediated Inflammatory Diseases, China-Japan Friendship Hospital, No. 2, Yinghua Dong Lu, Chaoyang District, Beijing, 100029, PR China Graduate School of Peking Union Medical College, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 9, Dongdan Santiao, Dongcheng District, Beijing, 100730, PR China c Beijing Key Laboratory of Diabetes Research and Care, Center for Endocrine Metabolism and Immune Diseases, Lu He Hospital, Capital Medical University, No. 82 South Xinhua Road, Tongzhou District, Beijing, 101149, PR China d Beijing University of Chinese Medicine, No. 11, Bei San Huan Dong Lu, Chaoyang District, Beijing, 100029, PR China

Received 3 February 2019; received in revised form 14 May 2019; accepted 19 July 2019

Abstract Formononetin has been reported to ameliorate hyperlipidemia and obesity, but its effect and mechanism of action in anti-non-alcoholic fatty liver disease (NAFLD) remain unclear. Lipophagy is a critical protective mechanism during steatosis development that results in the decomposition of lipid droplets through autophagy and the prevention of cellular lipid accumulation. This study aimed to investigate the beneficial role of formononetin in treating NAFLD and explore the mechanism of lipophagy in formononetin anti-hepatic steatosis effects. Formononetin treatment significantly ameliorated hepatic steatosis in HFD mice. Consistently, formononetin also reduced FFAs-stimulated lipid accumulation in HepG2 cells and primary mouse hepatocytes. Further analysis revealed that steatosis increased LC3B-II, a marker of autophagy, but caused blockade of autophagic flux associated with a lack of lysosomes. Treatment with formononetin promoted lysosome biogenesis and autophagosome-lysosome fusion, relieving the blockade in autophagic flux and further induced lipophagy. Mechanistically, formononetin activated adenosine monophosphate activated protein kinase (AMPK) and promoted subsequent nuclear translocation of transcription factor EB (TFEB), a key regulator of lysosome biogenesis. TFEB inhibition markedly abolished formononetin-induced lysosome biogenesis, autophagosome-lysosome fusion and lipophagy and concomitantly alleviated lipid accumulation. Formononetin improved hepatic steatosis via TFEB-mediated lysosome biogenesis, which provides new evidence regarding formononetin's anti-NAFLD effects. © 2019 Elsevier Inc. All rights reserved.

1. Introduction NAFLD is the main cause of chronic liver diseases worldwide and is characterized by overabundant hepatic lipid accumulation and aberrant serum lipid disorders [1]. Currently, lifestyle changes, diet adjustment and bariatric surgery are common treatments for NAFLD given the lack of effective FDA-approved drugs. These nonmedication methods are difficult for patients, and their effects are highly dependent on each individual. Thus, it is in urgent need for the

development of new drug therapies capable of alleviating hepatic steatosis with easy approaches. Formononetin (7-hydroxy-3(4-methoxyphenyl) chromone, Fig. 1A), is a natural isoflavone and widely enriched in various bead plants, such as licorice, pueraria, sandalwood, purple locust and red triaxial grass [2,3]. Extracts containing formononetin have been reported to reduce serum low-density lipoprotein-cholesterol (LDL-C) levels in a random double-blind controlled trial [4]. A formononetin analogue reduces adipose weight and levels of serum triglyceride (TG), total

Abbreviations: AMPK, adenosine monophosphate activated protein kinase; ALT, serum alanine aminotransferase; AST, aspartic acid transaminase; ATP6V1A, Vtype proton ATPase catalytic subunit A; AUC, area under the curve; BSA, bovine serum albumin; CQ, chloroquine; CPT1α, carnitine palmitoyltransferase-1α; DAPI, 4′,6-diamidino-2-phenylindole; FBS, fetal bovine serum; FFAs, free fatty acids; FMN, formononetin; GPO-PAP, glycerol phosphate oxidase-peroxidase 4-amino antipyrine and phenol; H&E, hematoxylin and eosin; HDL-C, high-density lipoprotein-cholesterol; HFD, high-fat diet; IPGTT, intraperitoneal administration of glucose; LDL-C, low-density lipoprotein-cholesterol; LAMP1, lysosome-associated membrane protein 1; ND, normal diet; ORO, oil red O; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1; PPARα, peroxisome proliferator-activated receptor-α; siRNA, small interfering RNA; S6K1, ribosomal protein S6 kinase β-1; TC, total cholesterol; TG, triglyceride; TFEB, transcription factor EB ⁎ Corresponding author. Tel.: +86 10 84205650; fax: +86 10 64227163. E-mail addresses: [email protected] (L. Peng), [email protected] (P. Li). https://doi.org/10.1016/j.jnutbio.2019.07.005 0955-2863/© 2019 Elsevier Inc. All rights reserved.

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Fig. 1. Formononetin mitigated HFD-induced hepatic steatosis. (A) The chemical structure of formononetin. (B-D) Body weight was recorded every week, and body weight gain and food intake were measured at the 16th week. (E, F) Liver index was calculated by liver weight (g)/body weight (g). (G) Liver triglyceride was measured using the GPO-PAP method. (F) Liver gross appearance of mice fed a HFD or ND with or without formononetin treatment at the 16th week. H&E staining, bar=50 μm, and Oil Red O staining, bar=50 μm. The data were expressed as the mean±S.E.M. ⁎⁎Pb.01 vs. ND group; #Pb.05, ##Pb.01 vs. HFD group.

cholesterol (TC), and LDL-C in hypercholesterolemic hamsters [5]. Formononetin also has antioxidant, anti-inflammatory, anti-apoptosis activities and estrogenic effects [3,6,7]. Although a few fatty acid βoxidation pathways in adipocytes have been investigated, the exact protective mechanism of formononetin against hepatic steatosis remains unclear. Macroautophagy is a dynamic process, including autophagosome sequestration, autophagosome-lysosome fusion, autolysosome degradation, and utilization of degradation products [8]. In recent years, intracellular lipid droplets have also been considered as substrates for macroautophagy via a process termed lipophagy [9]. Lipophagy disassembles lipids from lipid droplets and breaks down triglyceride into free fatty acids (FFAs) in the autolysosome, maintaining lipid homeostasis and avoiding intracellular lipid accumulation [10]. Proper quantities of lysosomes and normal functional lysosomes with active

hydrolytic enzymes in acidic cytoplasm are necessary for lipophagy, promoting autophagosome-lysosome fusion and autolysosome degradation at full capacity. However, chronic high-fat intake can damage lysosome biosynthesis, leading to insufficient lipophagy and cellular lipid accumulation [11]. Current general mechanism studies provided limited regulatory information for lysosome-related genes, but lysosomal genetic analysis identified a potential transcription factorbinding site for TFEB, which is responsible for the expression of most lysosomal genes and regulatory elements [12]. TFEB belongs to the microphthalmia-transcription factor E (MiT/TFE) subfamily of basic helix–loop–helix factors and is a key regulator of the autophagy-lysosome pathway [13,14]. Normally, TFEB is located in the cytosol and on the lysosome surface where it interacts with mTOR in an inactive phosphorylated form. TFEB translocates into the nucleus in response to

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nutrient depletion and stress conditions [15]. TFEB nuclear translocation accelerates lysosome biogenesis and lipophagy, subsequently fortifying free fatty acid β-oxidation in mitochondria [13]. Recent reports have demonstrated that TFEB overexpression promotes vast therapeutic effects in ethanol-induced or high-fat diet (HFD)-induced hepatic steatosis by rescuing lipid overload-induced lysosome dysfunction and enhancing lipolysis [16,17]. TFEB has become an idealized target for the treatment of NALFD through simultaneous enhancement of lipid decomposition in autolysosomes and fatty acid degradation in mitochondria [18]. In this study, we projected to study the beneficial effect of formononetin on hepatic steatosis and its underlying mechanism. We found that formononetin treatment prevented chronic HFDfed mice and FFAs-stimulated hepatic cells from hepatic steatosis via the recovery of TFEB nuclear translocation, subsequent lysosome biogenesis and lipophagy. This study afforded both in vivo and in vitro evidence for the potential application of formononetin in the diet for the prevention and treatment of NAFLD.

2.2. Animal experiments

2. Materials and methods

Human hepatoma HepG2 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Primary mouse hepatocytes were isolated from mice via collagenase IV perfusion through the inferior vena cava as previously described [19]. Hepatocytes were plated on Petri dishes coated with 0.1% collagen I. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml)/streptomycin sulfate (100 μg/ml). Cells were cultured in 5% CO2 at 37 °C, and the following experiments were performed when the cells reached approximately 70–80% confluence. Cells were incubated with FBS-free DMEM containing 1 mM free fatty acids (FFAs, oleate/palmitate, 2/1) and 1% bovine serum albumin (BSA) for 24 h to produce cellar steatosis. And FBS-free DMEM containing only 1% BSA were utilized to culture control cells in each experiment. Subsequently, cells were treated with formononetin at indicated concentrations and times. Compound C was used as an AMPK inhibitor. Cells were maintained in complete medium in all vitro experiments to avoid starvation-induced autophagy. Oleate and palmitate were dissolved as described previously [20]. For autophagosome-lysosome fusion and autolysosome measurement, we applied RFP-GFP-LC3 adenoviruses (GeneChem, Shanghai, China) to infect HepG2 cells according to the manufacturer's instruction. The LC3 ratio was examined using a confocal laser scanning microscopy after incubation with or without FFAs

2.1. Antibodies and reagents Formononetin (R5010), oleate (O1008), palmitate (P5585), chloroquine (CQ, C6628), Oil Red O (O0625), fetal bovine serum (10270), and LC3B antibody (L7543) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM (SH30021.01) was purchased from HyClone Laboratories (Logan, UT, USA). Compound C (S7306) was acquired from Selleck Chemicals (Houston, TX, USA). BODIPY 493/503 was purchased from Thermo Fisher Scientific (Waltham, MA, USA). TFEB antibody (MAB9170–100) was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against lysosome-associated membrane protein 1 (LAMP1) (15665), phosphorylated AMPK (2535S), and p62 (5114S) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against PGC1α (ab176328), V-type proton ATPase catalytic subunit A (ATP6V1A) (ab137574), Beclin1 (ab62557), phosphorylated ribosomal protein S6 kinase β-1 (p-S6K1) (ab2571) and carnitine palmitoyltransferase-1α (CPT1α) (ab128568) were purchased from Abcam (Cambridge, MA, USA). Antibodies against β-actin (sc-376,421) and peroxisome proliferator-activated receptor-α (PPARα) (sc9000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Eight-week-old C57BL/6 J mice weighing 23–25 g were obtained from Beijing HFK Bioscience (China). Following 1 week of acclimation, the mice were randomly divided into 4 groups (n=6, per group): normal diet (ND) group, normal diet with formononetin (ND + FMN) group, high-fat diet (HFD) group, and high-fat diet with formononetin (HFD + FMN) group. ND and HFD groups were fed with chow diet or HFD (containing 60% fat; Research Diets, Inc., New Brunswick, NJ, USA) for 16 continuous weeks. The ND + FMN and HFD + FMN groups were first fed a normal diet or high-fat diet for 2 weeks. Then, these two groups were gavaged with formononetin (100 mg/kg per day) for an additional 14 weeks. The ND and HFD groups were gavaged with the same volume of distilled water. Mice were maintained in a regular 12-h light–dark cycled period at a controlled temperature (22±2 °C) and relative humidity (65–75%) with water ad libitum. Body weight was recorded once a week and food intake was measured at the 16th week. All mice were sacrificed after fasting overnight and then were anesthetized with sodium barbiturates preceding sacrifice. Serum and tissue samples were collected rapidly for subsequent assays. Mice care and treatments were conducted according to the NIH Guiding Principles for the Care and Use of Laboratory Animals, and the protocol was approved by the Ethics Committee of the China-Japan Friendship Institute of Clinical Medical Sciences (Approval no.13005).

2.3. Cell culture and treatment

Fig. 2. Formononetin alleviated whole-body glucose metabolism and serum lipid disorders in mice fed a chronic high-fat diet. (A, B) Serum TG, TC, LDL-C, HDL-C, ALT and AST were assayed with an automatic analyzer. (C) After fasting 16 h, IPGTT was measured at 0, 15, 30, 60, 90, and 120 min; AUC was subsequently calculated. The data were expressed as the mean ±S.E.M. ⁎Pb.05, ⁎⁎Pb.01 vs. ND group; #Pb.05, ##Pb.01 vs. HFD group.

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Fig. 3. Formononetin ameliorated FFAs-stimulated hepatic steatosis in HepG2 cells and primary mouse hepatocytes. (A, D) HepG2 cell and primary mouse hepatocyte viability was determined with a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. (B, E) Intracellular triglyceride was measured using the enzyme method. (C, F) HepG2 cells and primary mouse hepatocytes were fixed with 4% paraformaldehyde in PBS, stained with 1 μg/ml BODIPY 493/503 for 15 min at 37 °C, and then incubated with DAPI at 22–25 °C; fluorescent images and intensity data were collected and calculated using a high-content screening system, bar=20 μm. The data were expressed as the mean±S.E.M. ⁎⁎Pb.01 vs. BSA group; #Pb.05, ##Pb.01 vs. FFAs group. and formononetin. The excited wavelength of red fluorescent protein-conjugated LC3 was 550–590 nm and that for green fluorescent protein conjugated LC3 was 395–495 nm. To determine whether nuclear TFEB is essential for lysosome biogenesis, HepG2 cells were transfected with nontargeting small interfering RNA (siRNA) and siRNA targeting TFEB (purchased from GenePharma, Shanghai, China) using the lipid-based transfection reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). The siRNA targeting human TFEB

sequences are presented below: sense, 5′-CAGGCUGUCAUGCAUUACATT-3′ and antisense, 5′-UGUAAUGCAUGACAGCCUGTT-3′. 2.4. Cell viability detection Cells (2×103 cells/well) were seeded in 96-well culture plates to allow cell adherence. To study cytotoxicity, cells were incubated with fresh complete

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Fig. 4. Steatosis-associated impairment of autolysosome degradation was restored by formononetin. (A, B) Western blot assay and relative intensity analysis of LC3B-II and p62 in HFDfed mice and FFAs-stimulated HepG2 cells with or without formononetin. (C) Immunofluorescent p62 puncta per cell (n=30) were counted in FFAs-stimulated HepG2 cells treated with or without formononetin for 24 h, bar=20 μm. (D) RFP-GFP-LC3 of HepG2 cells in response to formononetin treatment for 24 h; nuclei were stained with DAPI, bar=20 μm; the number of autolysosomes (red puncta) per cell (n=30) was counted. (E) Western blot assay and relative intensity analysis of LC3B-II in response to formononetin and CQ for 24 h. LC3B-II net flux was assessed by subtracting the amount of LC3B-II in the absence of CQ from the amount of LC3B-II in the presence of CQ for each of the conditions, and data are graphically displayed. (F) BODIPY 493/503 staining of lipid droplets in HepG2 cells treated with formononetin and CQ; nuclei were stained with DAPI, bar=20 μm. The data were expressed as the mean±S.E.M. ⁎Pb.05, ⁎⁎Pb.01 vs. BSA group or ND group; ##Pb.01 vs. FFAs group. medium containing 0, 5, 10, 20, 40 and 80 μM formononetin for 24 h. Then, cell viability was assayed by MTT. 2.5. Lipid droplet and lysosome staining After steatosis, cells were incubated with or without FFAs and formononetin for 24 h. Cells were fixed with 4% paraformaldehyde in PBS and stained with 1 μg/ml BODIPY 493/503 for 15 min at 37 °C. Cell images and fluorescence intensity were quantified using ImageXpress Micro XLS Widefield High Content Screening System (Molecular Devices, San Jose, CA, USA). For double fluorescent labeling, HepG2 cells were loaded with 100 nM LysoTracker Red DND-99 (Molecular Probes, USA) for 30 min at 37 °C. Subsequently, 1 μg/ml BODIPY 493/ 503 was added for 30 min at 37 °C, and fluorescent images were collected using a confocal laser scanning microscopy (Carl Zeiss AG, Oberkochen, Germany). Oil Red O staining and HE staining of liver sections were performed using standard protocols, and sections were observed under a light microscope (Olympus, Tokyo, Japan) [21]. Positive area of Oil Red O stained sections was calculated with Image J software.

were incubated with anti-p62 (1:200) antibody, anti-LAMP1 antibody (1:150) and anti-LC3B antibody (1:50) at 4 °C. On the second day, cells were incubated with Alexa Fluor® 488-conjugated goat anti-mouse antibody (1:200) or Alexa Fluor® 647-conjugated goat anti-rabbit antibody (1:200) for 30 min in the dark at 37 °C. Fluorescence of the colocalization of LC3B and LAMP1 was assessed using a confocal laser scanning microscopy (Carl Zeiss AG, Oberkochen, Germany). For immunoblotting, protein was extracted at 4 °C in RIPA Lysis Buffer (Solarbio, Beijing, China) containing protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). For TFEB detected with immunoblotting, nuclear and cytosolic fractions were separated by commercial kits according to the manufacturer's protocol (R0050 and BC3740, Solarbio, Beijing, China). The protein extracts were separated on the 8–12% polyacrylamide gel and then transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany) by electrophoresis. After sealing, the membrane was incubated with the primary specific antibodies at 4 °C overnight followed by secondary antibodies coupled with horseradish peroxidase. Protein strips were detected using an enhanced chemiluminescence method and quantified using ImageJ software.

2.6. Immunofluorescence and immunoblotting

2.7. Biochemistry measurement

For immunostaining, cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.3% Triton-X100 for 10 min. Cells were blocked with 5% BSA for 30 min and incubated with anti-TFEB antibody (1: 200). HepG2 cells

The triglyceride of cells and liver tissues were measured using a TG quantification kit (Jiancheng, Nanjing, Jiangsu, China) according to the manufacturer's instructions. Serum TC, LDL-C, HDL-C, alanine aminotransferase (ALT),

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Fig. 5. Formononetin alleviated hepatic steatosis by stimulating lysosome biogenesis. (A) Western blot assay and relative intensity analysis of nuclear TFEB and its downstream targets LAMP1, ATP6V1A, and PGC1α after HepG2 cells were incubated with formononetin for 24 h. (B) Representative immunofluorescence double staining of LC3B (showing autophagosome) and LAMP1 (showing lysosome) after HepG2 cells were incubated with formononetin for 24 h; nuclei were stained with DAPI, bar=10 μm; the number of colocalization of LC3B (showing autophagosome) and LAMP1 (showing lysosome) per cell (n=30) was counted. (C) Representative colocalization and double staining of BODIPY 493/503 plus lysotracker after HepG2 cells were incubated with formononetin for 24 h; nuclei were stained with DAPI, bar=10 μm; the number of colocalization of BODIPY 493/503 and lysotracker per cell (n= 30) was counted. (D, E) Immunofluorescence staining of TFEB and percentage of TFEB nuclear translocation in formononetin-treated HepG2 cells or primary mouse hepatocytes and matched BSA controls and FFAs controls; per field (n=6, 40x) was counted; bar=25 μm. (F) Western blot assay and relative intensity analysis of hepatic nuclear TFEB and its downstream targets, including LAMP1, ATP6V1A, PGC1α, LC3B-I/II, and p62, in HFD-fed mice with or without formononetin treatment. The data were expressed as the mean±S.E.M. ⁎Pb.05, ⁎⁎Pb.01 vs. BSA group; #Pb.05, ##Pb.01 vs. FFAs group or HFD group.

aspartic acid transaminase (AST), and fasting blood glucose (FBG) were assayed using an automatic analyzer (Abbott Diagnostics, Abbott Park, IL, USA).

3. Results

2.8. Statistical analyses

3.1. Formononetin mitigated hepatic steatosis and lipid disorders in HFD-fed mice

All experiments were repeated at least three times independently. Error bars represent S.E.M. The data were analyzed by Student's t-test or one-way analysis of variance with post hoc analysis, using SPSS version 19.0 (IBM Inc., Chicago, IL, USA). Here, Pb.05 and Pb.01 indicated statistical significance.

To examine the effects of formononetin on NAFLD, C57BL/6 mice were fed HFD or ND and treated with or without formononetin for 14 weeks. As shown in Fig. 1B–F, HFD feeding led to significant increases

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Fig. 6. TFEB knockdown weakened the improvement of lysosome biogenesis and autophagosome-lysosome fusion induced by formononetin. (A) Western blot assay and relative intensity analysis revealed that siRNA targeting TFEB interference reduced the upregulation of protein level in nuclear TFEB and its downstream targets, including LAMP1, ATP6V1A, and PGC1α, induced by formononetin in FFAs-stimulated HepG2 cells compared with matched controls. (B) Fluorescent assay of autolysosome ratio (RFP positive/GFP negative puncta ratio) after steatotic HepG2 cells were exposed to siRNA targeting TFEB and treated with or without formononetin for 24 h; nuclei were stained with DAPI, bar=20 μm; the number of autolysosomes (red puncta) per cell (n=30) was counted. (C) Representative immunofluorescence colocalization of LC3B (showing autophagosome) and LAMP1 (showing lysosome) after steatotic HepG2 cells were interfered with siRNA targeting TFEB, with or without formononetin; nuclei were stained with DAPI, bar=10 μm; the number of colocalization of LC3B (showing autophagosome) and LAMP1 (showing lysosome) per cell (n=30) was counted. (D) Representative colocalization and double staining of BODIPY 493/503 plus lysotracker after steatotic HepG2 cells were exposed to siRNA targeting TFEB treated with or without formononetin; nuclei were stained with DAPI, bar=10 μm; the number of colocalization of BODIPY 493/503 and lysotracker per cell (n=30) was counted. The data were expressed as the mean±S.E.M. #Pb.05, ##Pb.01 vs. FFAs group; &&Pb.01 vs. FFAs+FMN group.

in body and liver weight and hepatic TG accumulation compared with control ND feeding. Formononetin decreased body weight gain and hepatic TG accumulation in HFD-fed mice, while no significance was found in ND-fed mice. Food intake did not change between HFD-fed groups, indicating that the effects of formononetin on body weight and hepatic steatosis were not due to reduced food consumption. Morphologically, formononetin administration led to tighter and more ruddy liver gross appearance and a reduced number of ballooning degenerated hepatocytes and intracellular lipid droplets in HFD-fed mice (Fig. 1G). For whole-body lipid metabolism improvement, remarkably increased serum levels of TG, TC, and LDL-C in HFD-fed mice were partly reversed by formononetin administration, but the level of HDL-C was not impacted by formononetin intervention (Fig. 2A). As the liver plays a key role in energy balance and high-fat intake-induced NAFLD impairs liver function, we further assessed whether formononetin restores liver dysfunction in the HFD group. Serum ALT and AST levels were obviously decreased by formononetin supplementation, alleviating the adverse effects of HFD (Fig. 2B). HFD-fed mice developed insulin resistance during the formation of hepatic steatosis, which is characterized by abnormal intraperitoneal injection of glucose tolerance test (IPGTT) identified with higher level of the area under the curve (AUC) than ND-fed mice. After the treatment with formononetin, the AUC of HFD-fed mice decreases and the insulin resistance

was alleviated. Given that insulin resistance is closely related to the development of hepatic steatosis, the above IPGTT results suggested that formononetin has the potential to intervene the onset of hepatic steatosis, not only merely to mitigate hepatic steatosis (Fig. 2C). The residence time of formononetin in liver was from the 15th min to 4th h after gavaging formononetin (100 mg/kg per day), and formononetin was almost completely cleared from the liver at the 16th hour, excluding the potential damage of remaining drug (Fig. S1).

3.2. Formononetin ameliorated FFAs-stimulated lipid accumulation in HepG2 cells and primary mouse hepatocytes MTT results revealed that formononetin treatment at a concentration up to 20 μM had no effects on HepG2 cell and primary mouse hepatocyte viability (Fig. 3A and D and Fig. S2A and B). After 24 h stimulation of FFAs, cellular steatosis was successfully induced. Then, the level of intracellular TG in steatotic HepG2 cells were significantly alleviated by further incubation with formononetin for 24 h in a dose-dependent manner (Fig. 3B). And the fluorescent intensity of BODIPY 493/503-stained lipid droplets and intracellular TG were lowered by formononetin in steatotic HepG2 cells and primary mouse hepatocytes (Fig. 3C, E and F).

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Fig. 7. Inhibition of AMPK attenuated autophagy-associated activation of the mTOR pathway and Beclin 1 upregulation. (A) Western blot assay and relative intensity analysis revealed that AMPK inhibition via Compound C abolished the increased AMPK phosphorylation, S6K1 dephosphorylation and Beclin 1 expression induced by formononetin in FFAs-stimulated HepG2 cells compared with matched controls. (B) Western blot assay and relative intensity analysis of phosphorylation of AMPK and S6K1 as well as Beclin1 expression in HFD-fed mice treated with or without formononetin. The data were expressed as the mean±S.E.M. #Pb.05, ##Pb.01 vs. FFAs group or HFD group; &&Pb.01 vs. FFAs+FMN group.

3.3. Steatosis impaired autolysosome degradation in HFD-fed mice and FFAs-stimulated HepG2 cells Chronically sustained lipid challenges might strengthen autophagy pathways in hepatocytes and mice livers [22]. Western blot and semiquantitative analysis of integrated optical density revealed that the LC3B-II protein level dramatically increased. Moreover, p62, a marker of autophagosome-lysosome fusion blockade, was overtly increased in mice fed a HFD compared with mice fed a ND (Fig. 4A). These results indicate that pronounced lipid overload in mice may damage hepatic autolysosome degradation or autophagy flux. Consistently, FFAs-stimulated HepG2 cells exhibited increased LC3B-II levels and p62 expression compared with BSA-treated cells (Fig. 4B). The number of p62 dots determined by immunofluorescence staining in FFAs-stimulated HepG2 cells was also increased compared with BSA controls (Fig. 4C). Moreover, we generated RFPGFP-LC3 adenovirus-infected HepG2 cells to analyze autophagosome-lysosome fusion based on the fact that the green fluorescent protein (GFP) signal is quenched in acidic lysosomes. In contrast, the red fluorescent protein (RFP) signal is relatively stable. The ratio of red puncta (autolysosome with positive RFP and negative GFP signal) in BSA–treated cells was increased in FFAs-stimulated cells (Fig. 4D). These above results demonstrated that steatosis impaired hepatic autophagy degradation and alleviating the degradation or flux may help to alleviate lipid accumulation. 3.4. Formononetin ameliorated steatosis by stimulating autophagosomelysosome fusion in HepG2 cells Formononetin enhanced autophagy induction and autolysosome degradation in a dose-dependent manner, increasing LC3B-II expression and attenuating p62 protein levels in FFAs-stimulated HepG2 cells (Fig. S3). Similarly, the ratio of red puncta (positive RFP and negative GFP signal) in formononetin-treated cells was increased compared with that of BSA controls or FFAs controls (Fig. 4D), and the effects on autophagosome-lysosome fusion induced by formononetin require further characterization.

To monitor autophagy flux, we blocked autophagosomelysosome fusion by using chloroquine diphosphate (CQ, 10 μM), which is an inhibitor of lysosome function and autophagosome-lysosome fusion, and subsequently determined the turnover of LC3B-II (Fig. 4E). These results showed that formononetin treatment strengthened intracellular autophagy activity in FFAs-stimulated HepG2 cells. To determine whether formononetin-induced autophagy was related with its anti-hepatic steatosis effect, we employed CQ to block autophagy and assayed its impact on lipid droplets accumulation in HepG2 cells. As noted in Fig. 4F, formononetin-mediated reductions in intracellular BODIPY 493/ 503 fluorescence intensity were weakened by the presence of CQ, suggesting that formononetin-induced reductions in lipid droplet accumulation is associated with autophagy fluctuation in vitro. Thus, we hypothesized that formononetin promoted autophagy and alleviated hepatic steatosis mostly through autophagosome-lysosome fusion or autophagy flux improvement. 3.5. Formononetin alleviated steatosis by stimulating lysosome biogenesis Western blots revealed that nuclear TFEB and its downstream targets, LAMP1, ATP6V1A (V-type proton ATPase catalytic subunit A) and PGC1α, were obviously upregulated after formononetin incubation for 24 h in HepG2 cells (Fig. 5A). Immunofluorescence staining revealed that the level of colocalization of LC3B and LAMP1 in FFAs-stimulated HepG2 cells was lower than that of control cells, but the colocalization of LC3B and LAMP1 was increased upon treatment with formononetin, indicating the beneficial effect of formononetin in autophagosome-lysosome fusion (Fig. 5B). Lipophagy assayed by BODIPY 493/503 and lysotracker staining revealed a similar trend with colocalization of LC3B and LAMP1 in HepG2 cells (Fig. 5C). Given that TFEB modulates the expression of lysosomerelated genes, serves as the main regulatory factor for lysosome biogenesis and autophagosome-lysosome fusion pathway, we determined TFEB expression and its regulation on target genes in formononetin-treated hepatic cells. Immunofluorescent morphology results revealed that formononetin treatment increased the percentage of TFEB nuclear translocated HepG2

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Fig. 8. Schematic graph describing the mechanism of formononetin on hepatic steatosis. Formononetin activates AMPK and promotes nuclear translocation of TFEB, which is subsequently followed by autophagosome biogenesis, lysosome biogenesis, autophagosome-lysosome fusion and lipophagy, finally ameliorating lipid accumulation.

cells and primary mouse hepatocytes independent of FFAs (Fig. 5D and E). Meanwhile, formononetin stimulated nuclear translocation in steatotic HepG2 cells in Western blot assay (Fig.S4). Furthermore, formononetin upregulated hepatic nuclear translocation of TFEB and its downstream genes, including LAMP1, ATP6V1A and PGC1α, along with LC3B-II and p62 in mice fed a HFD compared with HFD controls (Figs. S5 and 5F). Formononetin also increased the hepatic protein level of fatty acid βoxidation genes, including PPARα and CPT1α, in steatotic HepG2 cells and mice fed a HFD (Fig. S6). Overall, these results indicated that formononetin strengthened the transcriptional activity of TFEB.

3.6. TFEB knockdown abated the improvement in lysosome biogenesis and autophagosome-lysosome fusion induced by formononetin We next performed instantaneous TFEB knockdown in HepG2 cells to validate the regulatory role of TFEB in lysosome-related gene expression. Under TFEB knockdown, LAMP1, ATP6V1A and PGC1α levels were notably decreased in formononetin-treated HepG2 cells (Fig. 6A). In addition, TFEB knockdown also weakened the formononetin-induced increase in the ratio of red puncta (positive RFP and negative GFP signal) and colocalization of LC3B and LAMP1, leading to lipophagy attenuation as determined by BODIPY 493/503 and lysotracker double staining (Fig. 6 B-D). Our results demonstrated that TFEB activation is essential for lysosome activation after formononetin therapy and suggested that TFEB activation is mediated by formononetin-induced lysosome biogenesis and autophagosome-lysosome fusion.

3.7. Inhibition of AMPK attenuated formononetin-induced TFEB nuclear translocation To identify whether formononetin-induced TFEB nuclear translocation involved AMPK, we used the specific AMPK inhibitor Compound C to measure its effect on the levels of phosphorylated AMPK and S6K1, a well-known substrate protein of mTORC1 [16]. AMPK inhibition abated the increase in AMPK phosphorylation and S6K1 dephosphorylation in formononetin-treated HepG2 cells. Beclin 1 exhibited similar changes as AMPK phosphorylation (Fig. 7A). Hepatic phosphorylation of AMPK and its direct target Beclin1 along with S6K1 in formononetin-treated mice fed a HFD was considerably increased compared with mice fed a HFD alone, which is consistent with the in vitro results (Fig. 7B). The results indicated that formononetin administration may promote hepatic autophagy activity through the AMPK/TFEB pathway. Thus, we summarized that formononetin restored autophagy activity and attenuated lipid accumulation mostly through the AMPK/TFEB lysosome biogenesis pathway in vitro. 4. Discussion The prevalence of NAFLD is estimated to reach 30% of the adult population and 70–80% of individuals who are obese and diabetic worldwide [23]. However, to date, the FDA has not yet approved any anti-NAFLD drugs [24]. Attempts to prevent and treat hepatic steatosis are increasingly focused on developing natural products derived from daily consumption food with minimal side effects and various health benefits. Formononetin, an O-methylated

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isoflavonoid that is rich in various beaded plants, has been reported to be effective against whole body lipid disorders [25]. Previous researchers demonstrated that formononetin and its analogues decreased lipid levels in adipocytes via enhancing fatty acid βoxidation via a peroxisome proliferator-activated receptordependent manner [25,26]. However, little information is known about the impact of formononetin on hepatic steatosis and its underlying mechanism. In this study, formononetin relieved hepatic steatosis in chronic HFD-fed mice and FFAs-stimulated hepatic cells and increased autophagy activity. Lipid droplets and lysosome double staining also revealed that formononetin promoted autolysosome phagocytosis of lipid droplets in steatosis hepatic cells, directly enhancing lipophagy. This study provides the first direct evidence for the anti-hepatic steatosis effect of formononetin and its lipophagy-related mechanism. Lipophagy is a critical protective mechanism to avoid lipid accumulation when organisms face excessive lipids. Portions of or entire lipid droplets are wrapped in the autophagosome and then decomposed into free fatty acids in the autolysosome [24]. Previous research suggests that when cells are exposed to high concentrations of FFAs or rodents subjected to a prolonged high-fat diet, autophagosome biogenesis was damaged, resulting in deficient lipolysis [27,28]. However, recent studies demonstrated that defects i n autophagosome-lysosome fusion impaired autophagy flux, leading to insufficient lipolysis and intracellular lipid accumulation. In contrast, lipid overload triggers autophagosome biogenesis [22,29]. This finding also explains why LC3B-II, an autophagosome marker, and p62, an autophagy substrate that indirectly reflects autophagy degradation inhibition, form stacks in livers of NAFLD patients, and this stacking positively correlates with disease severity [19]. In this research, LC3B-II and p62 levels were simultaneously increased in livers of HFD-fed mice and FFAs-stimulated hepatic cells compared with matched controls. This phenomenon is consistent with an autophagy flux blockade effect that resulted from the autophagy inhibitor chloroquine, which increases lysosomal pH and inhibits autophagosome-lysosome fusion. These effects lead to LC3B-II and p62 accumulation [23,30]. Subsequent formononetin intervention increased LC3B-II and decreased p62 in a dose-dependent manner in steatosis HepG2 cells, obviously counteracting autophagy flux blockade. Interestingly, formononetin induced steatosis remission in HepG2 cells was abolished by chloroquine, indicating formononetin could repair lipolysis defects due to insufficient autophagosome-lysosome fusion. Here, we identified that repairing autophagosome-lysosome fusion blockade represents a therapeutic target for steatosis and provides evidence that formononetin can improve hepatic steatosis via alleviating this blockade. Active lysosome biogenesis guarantees an appropriate number of lysosomes to fuse with autophagosomes that are synthesized in a compensatory manner under excessive lipid conditions. This process is regulated by the MiT/TFE subfamily, which regulates lysosomal genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network and including MITF, TFE3, TFEC, and TFEB [31]. TFEB is a master upstream regulatory element and upregulates approximately two-thirds of autophagy lysosome gene expression. Recent reports demonstrate that chronic ethanol consumption reduces lysosomes and induces more severe hepatic steatosis in TFEB knockout mice compared with matched controls [32]. These effects also promote TFEB nuclear translocation by pharmacologically inhibiting mTORC1 phosphorylationmediated lysosome biogenesis and lipid degradation and protecting against HFD-induced hepatic steatosis in mice [29]. Human studies also demonstrated that TFEB nuclear expression was reduced in human livers with NAFLD or non-alcoholic steatohepatitis (NASH) [15]. In this study, compared with ND-fed mice and BSA-treated hepatic cells, lipid overload prevents TFEB nuclear translocation and the expression of downstream lysosome-related genes, including LAMP1 and ATP6V1A [33]. Formono-

netin alleviated steatosis given that it partially reversed TFEB cytosolic retention and promoted nuclear translocation, reinforcing LAMP1 and ATP6V1 expression in vivo and in vitro. When TFEB was knocked down by gene suppression in steatotic HepG2 cells, the function of formononetin in promoting lysosome-related gene overexpression and increasing the number of lysosomes was impaired. These results suggest that formononetin could alleviate hepatic steatosis by rescuing insufficient autophagosome-lysosome fusion via TFEB-mediated lysosome biogenesis. Given that PGC1α upregulates hepatic mitochondrial biogenesis and mitochondrial fatty acid β-oxidation, another potential mechanism wherein formononetin acts against lipid overload-induced steatosis partially through TFEB-mediated PGC1α overexpression should not be excluded. The present study also indicated that formononetin has the potential to remove fatty acids generated from lipophagy and reduce lipotoxicity via enhancing expression of fatty acid β-oxidation related genes (PPARα and CPT1α). To further confirm the effect of formononetin on liver steatosis via autolysosome degradation, we still need to investigate more lipid metabolic changes in liver-specific TFEB knockout mice treated with vehicle or formononetin in the future. TFEB is regulated by multiple kinases, including mTORC1, PKCβ (protein kinase Cβ), PKCδ, GSK3β and Ca2+-dependent calcineurin. Currently, mTORC1-mediated negative regulation of TFEB activation is a well understood regulatory circuit [34]. AMPK is the main sensor of energy. AMPK regulates energy balance and metabolic stresses by controlling a variety of homeostatic mechanisms, including autophagy. Given that mTORC1 activity depends on AMPK status in steatotic cells, we assayed whether the mTORC1 and AMPK pathway was involved in the anti-steatosis effects of formononetin [28]. After the induction of steatosis, HepG2 cells were incubated with formononetin and Compound C. S6K1, a commonly established maker of mTORC1 activation, was highly rephosphorylated, and mTORC1 regained activity. These observations are consistent with previous studies that demonstrate that mTORC1 renews its activity in AMPK-abrogated cells. AMPK activation stimulated by formononetin was followed by increased levels of Beclin-1, which is a direct target of AMPK and positively regulates autophagosome biogenesis. Increased Beclin-1 levels were also reduced by Compound C. Overall, our data suggested that formononetin rescues autophagosome-lysosome fusion and improve lipophagy activity via regulating TFEB- and AMPKmediated autophagy in parallel. In response to nourishing nutrients, lipid oversupply also activates other AMPK-mediated transcription factors, such as silent information regulator 1 (SIRT1) and Forkhead box O3 (FOXO3), which could upregulate the transcription of various genes related with autophagic activity [35]. We hypothesize that AMPK may regulate SIRT, FOXO3 and other autophagic modulators, but this hypothesis requires further validation. Conclusively, when hepatic cells are cultured under excessive lipid conditions, defects in lysosome-restricted autophagosome-lysosome fusion, lipophagy and final lipolysis are noted, thus deteriorating intracellular lipid accumulation. Formononetin restores TFEB nuclear translocation, which is subsequently followed by lysosome biogenesis, autophagosome-lysosome fusion and lipophagy, alleviating lipid accumulation in an AMPK-dependent manner (Fig. 8). These results firstly explored the mechanisms of formononetin in treating hepatic steatosis, which affords therapeutic strategy for NAFLD. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgments This work was supported by National Natural Science Foundation of China (81620108031 and 81670758), Beijing Municipal Natural Science Foundation of China (7182147 and 7192191) and Capital's

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