Improvement of Lipid and Glucose Metabolism by Capsiate in Palmitic

Capsiate, a nonpungent ingredient of CH-19 Sweet, exhibits anti-obesity effects on animals and humans. This study investigated the effects and molecul...
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Improvement of Lipid and Glucose Metabolism by Capsiate in Palmitic Acid-Treated HepG2 Cells via Activation of AMPK/SIRT1 Signal Pathway Yufan Zang, Li Fan, Jihua Chen, Ruixue Huang, and Hong Qin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01831 • Publication Date (Web): 10 Jun 2018 Downloaded from http://pubs.acs.org on June 10, 2018

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Journal of Agricultural and Food Chemistry

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Improvement of Lipid and Glucose Metabolism by Capsiate in

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Palmitic Acid-Treated HepG2 Cells via Activation of

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AMPK/SIRT1 Signal Pathway

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Yufan Zang1,†, Li Fan1,†, Jihua Chen1, Ruixue Huang2, Hong Qin1,*

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1 Department of Nutrition Science and Food Hygiene, Xiangya School of

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Public Health, Central South University, 110 Xiangya Road, Changsha, Hunan

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Province, China, 410078

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2 Department of Occupational and Environmental Health, Xiangya School of

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Public Health, Central South University, 110 Xiangya Road, Changsha, Hunan

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Province, China, 410078

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*Corresponding author: Hong Qin, Tel: + 86 15974222668, E-mail:

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[email protected], Fax: + 86 0731 84805454.

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†These two authors contributed equally to this work.

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Abstract:Capsiate, a non-pungent ingredient of CH-19 Sweet, exhibits

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anti-obesity effects on animals and humans. This study investigated the effects

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and molecular mechanism of capsiate on lipid and glucose metabolism in

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PA-treated HepG2 cells. Results showed that compared with the PA-alone

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group, 100 μM capsiate inhibited lipid accumulation, decreased TG (0.0562±

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0.0142 vs 0.0381±0.0055 mmol/gprot, P = 0.024) and TC (0.1087±0.0037 vs

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0.0359±0.0059 mmol/gprot, P = 0.000) levels, while increased HDL-C level

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(0.0189±0.0067 vs 0.1050±0.0106 mmol/gprot, P = 0.000) and glycogen

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content (0.0065±0.0007 vs 0.0146±0.0008 mg/106 cells, P = 0.000) of

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PA-treated HepG2 cells. 100 μM Capsiate also up regulated the level of CD36

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(P = 0.000), phosphorylation of ACC (P = 0.034) and expression of CPT1 (P =

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0.013) in PA-treated HepG2 cells, leading to a positive enhancement of lipid

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metabolism. Meanwhile, 100 μM capsiate up regulated the levels of GLUT1,

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GLUT4, GK and phosphorylation of GS (P = 0.001, 0.029, 0.000, 0.045,

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respectively), down regulated PEPCK level (P = 0.001) to improve glucose

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metabolism in PA-treated HepG2 cells. Furthermore, the phosphorylation of

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AMPK and expression of SIRT1 in HepG2 cells were increased by 100 μM

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capsiate treatment (P = 0.001 and 0.000, respectively), while FGF21 level was

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decreased (P = 0.003). Most of these effects were reversed by pre-treatment

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with compound C, a selective AMPK inhibitor. Thus, capsiate might improve

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lipid and glucose metabolism in HepG2 cells by activating AMPK/SIRT1

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signaling pathway.

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Keywords: Capsiate; obesity; high fat; lipid metabolism; glucose metabolism

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INTRODUCTION

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Obesity is always accompanied by lipid and glucose metabolic disorders,

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and is a major cause for the development of hyperlipidemia and non-alcoholic

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fatty liver disease (NAFLD)1. As liver plays a key role in energy metabolism,

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extensive efforts have been focused on the treatment of the hepatic lipid and

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glucose metabolic disorders associated with obesity. Currently, although

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several therapeutic agents of NAFLD have been assessed, there are no

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pharmacotherapies specifically approved for NAFLD management2, 3. Hence,

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finding novel phytochemicals that extracted from normal food, which could

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target both the hepatic lipid as well as glucose metabolism disorder, is of

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significant interest.

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Recently, health benefits associated with the consumption of capsiate,

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which is extracted from sweet pepper Capsicum CH-194, have been well

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reported. It has been demonstrated that capsiate could relieve inflammation5

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and attenuate insulin resistance 6, both of which are metabolic disorders

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associated with obesity. Currently, capsiate has been shown to serve as

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promising therapies for obesity, such as promoting white fat “browning” and

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stimulating energy consumption7-10. In addition, our previous study and some

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other studies suggested that capsiate could improve lipid metabolism and

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promote the use of glucose in liver of obese rodents 6, 11, 12. Some of these

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studies demonstrated that the metabolic homeostasis effect of capsiate partly

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dependent on the activation of transient receptor potential vanilloid subtype 1

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(TRPV1) receptor7, 9. However, specific mechanisms of capsiate remain largely

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unexplored. In view of the multiple physiological effects, we supposed that

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capsiate could relieve lipid and glucose metabolic disorders of NAFLD via

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more complicated signaling pathways other than TRPV1. It is accepted that the activation of adenosine monophosphate-activated

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protein kinase (AMPK), the energy sensor protein, is an important step in

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reducing lipid accumulation in liver13. Once activated, AMPK modulates

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hepatic energy metabolism by multiple mechanisms such as enhancing fatty

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acid oxidation, inhibiting lipid synthesis as well as repressing

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gluconeogenesis14. Sirtuin 1 (SIRT1) is another key regulator, which involved

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in hepatic lipid and glucose metabolic homeostasis by stimulating various

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downstream signaling proteins15. In this regard, finding some phytochemical to

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activate AMPK and SIRT1 is a sensible strategy in the treatment of NAFLD 16,

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17

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. The present study was the first to examine if capsiate could regulate the

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hepatic lipid and glucose metabolism simultaneously under high fat

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environment in vitro. We also sought to ascertain the exact mechanism of

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capsiate on energy metabolism. The major findings of this study were that

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capsiate reduced lipid accumulation and increased glycogen synthesis of

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HepG2 cells that treated with palmitic acid (PA), and the AMPK/SIRT1

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signaling pathway was critical for these observed effects.

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MATERIALS AND METHODS

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Chemicals. Capsiate (97.3 % pure) was purchased from Sigma-Aldrich (St.

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Louis, MO, USA). PA was purchased from Regent Science (Shenzhen, China).

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HepG2 cells were purchased from Peking Union Cell Center (Beijing, China).

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Oil red O was purchased from Solarbio Science & Technology Co., Ltd.

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(Beijing, China). Triglyceride (TG), total cholesterol (TC), high-density

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lipoprotein cholesterol (HDL-C) and glycogen assay kits were purchased from

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Jiancheng Bioengineering Institute (Nanjing, China). MTT assay kit and the

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Goat anti-Rabbit IgG antibody combined with horseradish peroxidase were

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purchased from Ding Guo Changsheng Biotechnology Co., Ltd. (Beijing,

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China). Antibodies against cluster of differentiation 36 (CD36, #A1470),

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carnitine palmityl transferase 1 (CPT1, #A5037), glucose transporter 1 (GLUT1,

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#A6982), glucose transporter 2 (GLUT2, #A9843), glucose transporter 4

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(GLUT4, #A7637), glucokinase (GK, #A6293), phosphoenolpyruvate

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carboxykinase (PEPCK, #A4005), SIRT1 (#A0230) and β-actin (#AC026) were

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purchased from ABclonal (Boston, MA, USA). Antibodies against acetyl CoA

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Carboxylase (ACC, #3676), phospho-Acetyl-CoA Carboxylase (p-ACC,

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#11818), AMPKα (#5831) and phospho-AMPKα (p-AMPKα, #2535) were

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purchased from Cell Signaling Technology, Inc . (Boston, MA, USA). Antibodies

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against glycogen synthase (GS, #ab40810), phospho-GS (p-GS, #ab81230)

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and fibroblast growth factor 21 (FGF21, #ab171941) were purchased from

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Abcam Inc. (Cambridge, UK). Compound C was purchased from MCE (NJ,

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USA).

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PA Preparation. PA was prepared according to the method previously

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described by Qin et al18. Briefly, PA was dissolved in 100mM NaOH solution at

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70 ℃, and mixed with 10% (w/v) fatty acid-free bovine serum albumin (BSA)

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at 55 ℃. The concentration of the stock PA solution was 5 mM. Then the liquid

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was membrane-filtered and diluted 1:20 (v/v) with Dulbecco’s modified Eagle’s

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medium (DMEM) to obtain 0.25 mM PA.

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Cell Culture. HepG2 cells were cultured in 4.5 g/L glucose DMEM with 10%

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(v/v) fetal bovine serum (FBS) and 1% Penicillin-Streptomycin Solution (100

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U/mL penicillin and 100 μg/mL streptomycin). After reaching 80% confluence,

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the cells were treated with or without 0.25mM PA for 24h. Then the cells which

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exposed to PA were treated with capsiate at different concentrations (0, 25, 50,

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and 100 μM) for 24h. In the experiments with the AMPK inhibitors, HepG2 cells

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were pre-treated with 10 μM compound C for 1 h prior to capsiate treatment.

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MTT Assay. MTT assay was used to measure cell viability. HepG2 cells were

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seeded at a density of 6.0 × 103 cells/well in 96-well plates and cultured

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overnight. Then cells were treated with different concentrations of PA (0, 0.125,

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0.25, 0.5 and 1.0 mM) for 48 h, or different concentrations of capsiate (0, 12.5,

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25, 50, 100, 200 and 400 μM) for 24 h. For further studies, HepG2 cells were

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also treated with 0.25 mM PA for 24h and then co-incubated with different

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concentrations of capsiate (0, 12.5, 25, 50, 100, 200 and 400 μM) for an

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additional 24 h. And then, 20 μL of MTT solution was added to each well for 4 h

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at 37 ℃. Next, the supernatants was removed, the formazan crystals were

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dissolved in 150 μL DMSO. The absorbance values were measured at 570 nm

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by a microplate reader (Power Wave XS2; BioTek Instruments, Inc., VT).

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Lipid Content Assays. Lipid accumulation was detected by oil red O staining

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which performed according to a slightly modified method previously described

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by Weston et al19: after fixing in 4% paraformaldehyde for 30 min, the cells

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were washed in PBS and then stained with freshly prepared oil red O for 30

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min, following rinsing with 60% isopropanol and distilled water. Subsequently,

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the lipid droplets were observed under an inverted microscope. The contents

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of TG and TC were detected by GPO-PAP method, and the content of HDL-C

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was measured by double reagent method using commercially available kits.

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The content of each index was measured by a microplate reader and

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expressed as mmol / gprot.

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Glycogen Content Assay. Each group of cells was counted, and then the

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glycogen content of HepG2 cells was detected by anthrone-sulfuric acid

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method using a glycogen assay kit. Briefly, HepG2 cells were digested and

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centrifuged at 156 ×g for 10 min, and the precipitates were transferred to

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glass tubes with 0.225 mL alkaline liquor. Samples were incubated in boiling

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water for 20 min, and then 0.1 mL hydrolyzate was diluted by distilled water

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and 2 mL color-substrate solution, according to the instruction. Subsequently,

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samples were incubated in boiling water for 5 min. Thereafter, the OD value of

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each group was determined at 620 nm by an ultra-micro spectrophotometer

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(Implen GmbH, Schatzbogen, Germany). The cell total glycogen content was

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calculated and expressed as mg /106 cells.

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Western Blot Analysis. The cells were lysed in RIPA buffer and centrifuged at

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14000 ×g for 12 min to obtain the supernatant. Then the total protein content

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of each group was measured by an ultra-micro spectrophotometer.

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Subsequently, volumes of extracted protein samples were calculated

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according to 40 μg per group, and then the samples were separated by

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SDS-PAGE and transferred 2 h to polyvinylidene difluoride (PVDF)

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membranes. Especially, ACC and p-ACC were transferred 5 h to PVDF

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membranes. After blocking with 5% skim milk powder or 3% BSA in TBST, the

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membranes were incubated with ACC (1:1000), p-ACC (1:1000), CPT1

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(1:1000), CD36 (1:1000), GLUT1 (1:1000), GLUT2 (1:1000), GLUT4 (1:1000),

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GK (1:1000), PEPCK (1:1000), GS (1:10000), p-GS (1:10000), AMPKα

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(1:1000), p-AMPKα (1:1000), SIRT1 (1:1000) or FGF21 (1:1000) primary

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antibodies overnight at 4 ℃. And β-actin (1:300000) was used as a reference

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protein. After being washed in TBST, the membranes were incubated with the

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goat anti-rabbit IgG antibody (1:6000, Ding Guo Changsheng Biotechnology

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Co., Ltd., Beijing, China) for 1 h at room temperature. Protein bands were

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detected using the chemiluminescence imager (Tanon-5500, Tanon Science &

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Technology Co., Ltd. Shanghai, China) and the intensities were assessed by

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Autogel software. The expression levels of proteins were analyzed by

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semi-quantitative method.

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Statistical Analysis. All analyses were done with SPSS 18.0 software (SPSS

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Inc., Chicago, IL, USA). The data were shown as means ± SD (n = 3).

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Differences among groups were calculated by one-way analysis of variance

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(ANOVA) and LSD post-hoc test (more than two groups), or independent

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samples t-test (two groups). P < 0.05 was considered to indicate a statistically

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significantly difference.

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RESULTS

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Effects of PA and Capsiate on HepG2 Cell Viability. For cellular toxicity,

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the MTT assay was performed to ensure that treatment with PA and capsiate

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did not affect cellular viability. Neither the treatment with 0.25 mM of PA for 48

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h nor the treatment with 0–400 μM of capsiate for 24 h inhibited the growth of

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HepG2 cells (Fig. 1A, 1B). In addition, the viability of HepG2 cells which

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exposed to PA did not be reduced after treatment with 0–100 μM of capsiate,

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but it could be affected after treatment with 200 and 400 μM of capsiate (Fig.

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1C). Based on these results, 25, 50 and 100 μM of capsiate were selected for

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our experiments.

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Effects of Capsiate on Lipid Accumulation in PA-Treated HepG2

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Cells. Treatment with 0.25 mM PA led to a dramatic increase of lipid droplets in

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HepG2 cells compared with the normal control group (Fig. 2A, 2B). After

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treating with capsiate, the cells contained significantly less lipid droplets (Fig.

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2C, 2D, 2E). The result indicated that capsiate could effectively reduce the lipid

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accumulation in PA-treated HepG2 cells.

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Effects of Capsiate on TG, TC and HDL-C Contents in PA-Treated

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HepG2 Cells. Compared with the control group, the TG and TC contents were

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significantly increased (0.0246±0.0045 mmol/gprot vs 0.0562±0.0142

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mmol/gprot, P = 0.001 and 0.0086±0.0049 mmol/gprot vs 0.1087±0.0037

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mmol/gprot, P = 0.000, respectively) in 0.25 mM PA-treated HepG2 cells. To

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verify the improvement effect of capsiate on lipid metabolism, the cells which

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exposed to PA were treated with capsiate at different concentrations. Results

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showed that compared with the PA-alone group, capsiate effectively

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decreased TG (0.0506±0.0043, 0.0452±0.0087, 0.0381±0.0055 mmol/gprot

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at 25 μM, 50 μM and 100 μM, respectively) and TC (0.0741±0.0064, 0.0255

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±0.0042, 0.0359±0.0059 mmol/gprot at 25 μM, 50 μM and 100 μM,

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respectively), while increased HDL-C (0.0540±0.0076, 0.0798±0.0226,

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0.1050±0.0106 mmol/gprot at 25 μM, 50 μM and 100 μM, respectively) in

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PA-treated HepG2 cells (Fig. 3A, 3B, 3C). These findings suggested that

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capsiate could improve lipid metabolism in PA-treated HepG2 cells.

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Effects of Capsiate on Glycogen Content in PA-Treated HepG2 Cells.

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Compared with the control group, glycogen content in the 0.25 mM PA-treated

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group was significantly decreased (0.0185±0.0020 mg/106 cells vs 0.0065±

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0.0007 mg/106 cells, P = 0.008). And compared with the PA-alone group,

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capsiate effectively increased glycogen content (0.0090±0.0014, 0.0110±

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0.0018, 0.0146±0.0008 mg/106 cells at 25 μM, 50 μM and 100 μM,

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respectively) in PA-treated HepG2 cells (Fig. 3D), which suggested that

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capsiate promoted glycogen synthesis, supporting the hypothesis that

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capsiate treatment improved glucose metabolism in PA-treated HepG2 cells.

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Effects of Capsiate on the Expressions of Lipid Metabolism-Related

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Proteins in PA-treated HepG2 Cells. To determine how capsiate improves

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lipid metabolism, we measured the expression of CD36, phosphorylation of

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ACC and expression of CPT1, which are essential for lipid uptake, lipogenesis

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and β-oxidation, respectively. After treating with 0.25 mM PA, the HepG2 cells

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showed a markedly increase in CD36 (P = 0.002) and decrease in

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phosphorylation of ACC (P = 0.017) and expression of CPT1 (P = 0.012). The

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treatment with capsiate further increased CD36 (P = 0.000 at 100 μM) to up

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regulate the fatty acid translocase (Fig. 4A), but it increased the ratio of

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phosphorylation of ACC (P = 0.034 at 100 μM) to inactivate this lipid synthesis

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enzyme (Fig. 4B). In the meantime, capsiate caused the ascent of CPT1 (P =

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0.013 at 100 μM), which is responsible for the β-oxidation (Fig. 4C). These

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results indicated that capsiate improved lipid metabolism by inactivating ACC

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as well as increasing CD36 and CPT1 in PA-treated HepG2 cells.

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Effects of Capsiate on the Expressions of Glucose Metabolism-Related Proteins in PA-Treated HepG2 Cells. For further

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understanding of the effects of capsiate on glucose metabolism in high fat

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environment, we measured the expressions of glucose metabolism-related

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proteins, such as GLUT1, GLUT2, GLUT4, GK, PEPCK and GS. In high fat

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environment, capsiate decreased GLUT2 (P = 0.029 at 100 μM), the main

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isoform of glucose transporters in liver, and increased GLUT1 and GLUT4 (P =

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0.001 and 0.029 at 100 μM, respectively), which are responsible for promoting

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the diffusion of glucose (Fig. 5A, 5B, 5C). Meanwhile, the treatment with

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capsiate increased GK level (P = 0.000 at 100 μM) to activate this glycolysis

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rate-limiting enzyme, as well as decreased PEPCK level (P = 0.001 at 100 μM)

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to inactivate this gluconeogenesis rate-limiting enzyme (Fig. 5D, 5E). However,

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the intervention of capsiate also resulted in an increase of phosphorylation of

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GS (P = 0.045 at 100 μM) to inactivate the enzyme for glycogen synthesis (Fig.

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5F). Taken together, these results indicated that capsiate regulated glucose

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metabolism by altering levels of GLUT1, GLUT2, GLUT4, GK, PEPCK as well

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as phosphorylation of GS in PA-treated HepG2 cells.

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Effects of Capsiate on the Expressions of Key Metabolic Regulators

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in HepG2 Cells. AMPK, an upstream kinase of ACC, CPT1, GLUT4, GK and

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PEPCK, is an important sensor in energy metabolism. SIRT1 also plays a

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major role in regulating energy metabolism and partially involved in the AMPK

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signaling pathway. To further investigate the mechanism by capsiate exerted

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its metabolic regulatory activity, the phosphorylation status of AMPK and the

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level of SIRT1 in HepG2 cells were examined. We found that capsiate

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effectively increased the phosphorylation of AMPKα (P = 0.001 at 100 μM) and

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the level of SIRT1 (P = 0.000 at 100 μM) in PA-treated HepG2 cells (Fig. 6A,

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6B), indicating that capsiate might improve energy metabolism via the

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activation of AMPK/SIRT1 pathway.

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We also measured the phosphorylation of AMPKα and the level of SIRT1

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in PA-free HepG2 cells with or without 100 μM capsiate. We found that

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capsiate could significantly increase the phosphorylation of AMPKα (P = 0.018)

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and the expression of SIRT1 (P = 0.006) in HepG2 cells even in PA-free

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environment (Fig. 6C, 6D). These results suggested that capsiate activated

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AMPK/SIRT1 signaling pathway in a PA-independent manner.

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Moreover, FGF21 has attracted significant attention as a novel metabolic

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regulator recently, so we also assessed the FGF21 level in HepG2 cells.

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Interestingly, FGF21 was significantly increased (P = 0.002 at 100 μM) in

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HepG2 cells by 0.25 mM PA and was decreased (P = 0.003 at 100 μM) after

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treating with capsiate (Fig. 6E). This phenomenon perhaps due to a

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self-regulation of liver caused by changes in lipid and glucose metabolism

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status20 .

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Effects of Compound C on Capsiate-Treated HepG2 Cells in High Fat

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Environment. To confirm whether the effects of capsiate on lipid and glucose

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metabolism are mediated by AMPK activation, compound C, a specific inhibitor

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of AMPK, was used. Compared with the PA + capsiate group,

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capsiate-induced decreases of lipid droplets, TG and TC contents in HepG2

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cells were abolished (0.0571±0.0073 mmol/gprot vs 0.1197±0.0051

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mmol/gprot, P = 0.000 and 0.0476±0.0051 mmol/gprot vs 0.0911±0.0070

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mmol/gprot, P = 0.000, respectively) by pre-treatment with compound C (Fig.

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7A, 7B). In addition, compared with the PA + capsiate group, capsiate-induced

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increase of glycogen content was decreased (0.0078±0.0002 mg/106 cells vs

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0.0010±0.0004 mg/106 cells, P = 0.000) with compound C (Fig. 7C).

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Meanwhile, the phosphorylation of AMPKα and the expression of SIRT1 in

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PA-treated HepG2 cells incubated with capsiate in the presence of compound

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C were significantly decreased (P = 0.000 and 0.001, respectively), and the

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expression of FGF21 was increased (P = 0.048) (Fig. 7D). Furthermore, the

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increases of phosphorylation of ACC, GLUT1, GLUT4, GK and

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phosphorylation of GS induced by capsiate in PA-treated HepG2 cells were all

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reversed (P = 0.002, 0.000, 0.011, 0.001 and 0.000, respectively) in the

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presence of compound C. However, pre-treatment with compound C did not

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affect capsiate-induced increase of CD36 (P = 0.621) and decreases of

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GLUT2 (P = 0.409) in HepG2 cells in high fat environment, and it even further

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decreased PEPCK (P = 0.012) (Fig. 7E, 7F). Taken together, these above

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results indicated that capsiate improved lipid and glucose metabolism in

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PA-treated HepG2 cells at least partly via activation of AMPK.

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DISCUSSION

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In our current in vitro study, the important findings were that capsiate

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could reduce lipid accumulation, decrease TG and TC, increase HDL-C and

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glycogen of HepG2 cells in high fat environment. These significant effects of

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capsiate were mediated by the increases of CD36, phosphorylation of ACC,

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CPT1, GLUT1, GLUT4, GK, phosphorylation of GS, as well as by the

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decreases of PEPCK in PA-treated HepG2 cells. With further study on the

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mechanism of capsiate, we focused on AMPK and SIRT1, both of which were

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energy regulating factors in hepatic cells 14, 15. The novelty of our study was that

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we elucidated that capsiate regulated energy metabolic homeostasis via

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activation of AMPK/SIRT1 signal pathway.

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Liver is one of the primary tissues of energy metabolism. Obese

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associated high fat environment always leads to metabolic disorders in liver,

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such as abnormal lipid accumulation, insulin resistance and glycogen

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reduction21. Therefore, improving hepatic energy metabolism is of great

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meaning in the treatment of obesity and obesity-related diseases. Since

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HepG2 cells displayed many of the genotypic features of normal human

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hepatocytes22 and were used wildly to model hepatic function in vitro 23, 24, in

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this study, we simulated the high fat environment by treating HepG2 cells with

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PA25. Consistent with previous studies 26, our current study showed that PA

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could induce lipid accumulation and glycogen reduction in HepG2 cells . Herein,

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we found that capsiate, a non-pungent compound extracted from CH-19 Sweet,

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could improve both lipid metabolism and glucose metabolism in PA-treated

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HepG2 cells.

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Then we focused on the mechanism by which capsiate modulated energy

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metabolism in HepG2 cells. We first evaluated the effects of capsiate on lipid

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metabolism of HepG2 cells in high fat environments, which primarily

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determined by the uptake and oxidation of free fatty acid and the lipid

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synthesis. The present result was in agreement with other studies that in high

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fat environment, expression of CD36 was increased to enhance fatty acid

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uptake in liver27, phosphorylation of ACC and expression of CPT1 were

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significantly decreased11, 28. Our study demonstrated that capsiate further

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increased expression of CD36, increased phosphorylation of ACC and level of

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CPT1 in PA-treated HepG2 cells. CD36 is a class B scavenger receptor that

327

binds lipids and enables their transport into cells followed by next steps of

328

lipids metabolisms, such as β-oxidation29. The further increase of CD36 by

329

capsiate indicated that capsiate might promote the utilization of fatty acid in

330

HepG2 cell. ACC is a key lipid synthesis enzyme that could be inactivated by

331

phosphorylation. Inactivation of ACC would reduce the synthesis of malonyl

332

coenzyme A, a CPT1 inhibitor, thereby activating the β-oxidation essential

333

enzyme CPT1, which participates in lipid oxidation and fat reduction30. By

334

regulating ACC and CPT1, capsiate inhibited lipid synthesis and promoted

335

β-oxidation to correct the lipid metabolism disorder in HepG2 cells, which was

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validated by the inhibition of lipid accumulation, the reduction of TG and TC, as

337

well as the increase of HDL-C.

338

We next determined if capsiate improved glucose metabolism of

339

PA-treated HepG2 cells. The liver regulates glucose homeostasis mostly by

340

maintaining balance between glycogenesis and glycolysis or gluconeogenesis.

341

Glycogen is an indicator of glucose metabolism, and the increase of liver

342

glycogen accumulation could attenuate lipid and glucose metabolic disorders

343

in obese mammals31. Our study found that capsiate reversed the reduction of

344

glycogen in PA-treated HepG2 cells, which indicated that capsiate alleviated

345

the disorder of glucose metabolism. Then, we observed the protein levels of

346

key glucose transporters, GLUT1, GLUT2 and GLUT4. GLUT2 is the main

347

mediator of glucose uptake and export across the hepatocyte plasma

348

membrane, which can increase hepatic glucose output in parallel with enhance

349

gluconeogenesis in glucose metabolic disorder status32. In liver, once glucose

350

metabolism disorder arises in the obese state, GLUT2 expressions would

351

increase33, 34. We observed the reduction of GLUT2 by capsiate in high fat

352

environment, which confirmed the beneficial effect of capsiate on hepatic

353

glucose homeostasis. GLUT1 and GLUT4 are glucose transporters

354

responsible for promoting the diffusion of glucose. In our study, GLUT1 and

355

GLUT4 were up regulated by capsiate and might be the major transporters of

356

hepatic glucose uptake in high fat environment. Furthermore, the results of

357

present research suggested that capsiate facilitated glucose utilization by

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raising glycolytic enzyme GK, and inhibited glucose synthesis by reducing

359

gluconeogenic enzyme PEPCK in HepG2 cells. In addition, GS is a key

360

enzyme for glycogen biosynthesis which could be inactivated by

361

phosphorylation. Of interest, we found that even though capsiate up regulated

362

the phosphorylation of GS to inactivate GS, the intracellular glycogen content

363

was increased instead of being decreased. This phenomenon indicated that

364

capsiate might promote glycogen synthesis in some other ways, such as

365

activating AMPK. Other studies have reported that although AMPK caused GS

366

phosphorylation, it was capable of stimulating glycogen synthesis35. Above

367

results forced us to think about the possible mechanism by which capsiate

368

improved hepatocyte energy metabolism was AMPK-mediated.

369

AMPK is a central switch for regulating liver energy homeostasis, and

370

activating AMPK is of great significance in the treatment of obesity and

371

obesity-related metabolic disorders13, 36. We know that this protein can be

372

activated by the phosphorylation of its α-subunit, and then promotes the

373

phosphorylation of ACC and the expressions of CPT1, GLUT1, GLUT4, GK,

374

and inhibits the expression of PEPCK, finally improves the metabolism of lipid

375

and glucose37-40. Therefore, we hypothesized that AMPK was likely to be

376

targeted by capsiate. As expected, capsiate increased the phosphorylation of

377

AMPKα in PA-treated HepG2 cells, and even under normal condition, capsiate

378

also showed the capacity on activating AMPK. These results suggested that

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capsiate activated AMPK to regulate lipid and glucose metabolism in HepG2

380

cells.

381

Furthermore, previous studies mentioned that phosphorylation of AMPKα

382

could activate SIRT1 directly41, and with regard that SIRT1 also plays an

383

important role in the regulation of lipid and glucose metabolism 42, 43, we

384

speculated that if SIRT1 was another possible target of capsiate. Thus, we

385

detected the SIRT1 expression of HepG2 cells in this study. In line with

386

previous observation44,there was lack of change in SIRT1 whether the HepG2

387

cells were treated with PA or not. However, after treated by capsiate, the cells

388

expressed significant higher SIRT1. We therefore demonstrated for the first

389

time that, capsiate could simultaneously increase the phosphorylation of

390

AMPK and the expression of SIRT1 to improve lipid and glucose metabolism in

391

HepG2 cells.

392

In addition, FGF21, a novel metabolic stress hormone mainly secreted by

393

liver20, 45, 46, was increased in NAFLD and considered as an independent

394

predictor of fatty liver disease 47-49. The present study showed that FGF21 of

395

HepG2 cells in high fat environment were reduced by capsiate. Nevertheless,

396

some previous studies reported that the trend of FGF21 changed resembled to

397

AMPK/SIRT1, which might be due to the role of FGF21 as an upstream

398

activator to AMPK/SIRT1 for high fat environment adaptations

399

we found that the activation of AMPK/SIRT1 was increased by capsiate rather

400

than decreased with the reduction of FGF21. We assumed that the metabolic

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. However,

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401

disorders have been attenuated by capsiate, therefore, the hepatocyte had no

402

more need for releasing FGF21 to adapt high fat environment. Further

403

research of effects of capsiate on FGF21 and metabolic disorders must be

404

compelling.

405

To further confirm whether the effects of capsiate were mediated by

406

activation of AMPK, we used compound C to inhibit AMPK. We found that

407

changes of most indexes induced by capsiate, such as phosphorylation of

408

ACC, GLUT1, GLUT4, GK and phosphorylation of GS, were reversed by

409

compound C. Furthermore, the expression of SIRT1 was also significantly

410

decreased by compound C, which partly demonstrated that SIRT1 might be

411

located downstream of AMPK. These phenomena suggested that capsiate

412

exerted its beneficial metabolic effects by the activation of AMPK/SIRT1

413

pathway in PA-treated HepG2 cells.

414

However, we also found that pre-treatment with compound C did not affect

415

capsiate-induced increase of CD36 and decrease of GLUT2, and it even

416

further reduced PEPCK in high fat environment. These phenomena suggested

417

that AMPK/SIRT1 was one of the targets of capsiate, and more work is needed

418

to distinguish the other mechanisms co-existed in the regulation of energy

419

metabolism by capsiate.

420

In conclusion, this study reported the novel finding that capsiate improved

421

cellular lipid and glucose metabolism in HepG2 cells in high fat environment,

422

which may be associated with the activation of AMPK/SIRT1 pathway. Since

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capsiate is hydrolysed and essentially absorbed as vanillyl alcohol in vivo,

424

which held the same biological properties of capsiate52, the outcome of the

425

current study indicated an effective therapeutic approach for treatment of

426

obesity-related hepatic disease. We proposed that better understanding of

427

capsiate could open up possibilities to manipulate energy expenditure in

428

patients with obesity and obesity-related metabolic diseases by new drugs or

429

even with dietary supplementation.

430

ACKNOWLEDGMENT

431

We thank Prof. Ji-hua Chen for helpful assistance with the experiments. We

432

also thank Prof. Rui-xue Huang for valuable advice.

433

AUTHOR INFORMATION

434

Corresponding Author

435

*Phone: +8615974222668. E-mail: [email protected]. Fax: + 86 0731

436

84805454.

437

ORCID

438

Hong Qin: 0000-0002-4578-5118

439

Funding

440

This study was supported by National Natural Science Foundation of China

441

(No.81302421), Natural Science Foundation of Hunan Province (No.

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2018JJ2550) and Fundamental Research Funds for the Central Universities of

443

Central South University (1053320180830).

444

Notes

445

The authors declare no competing financial interest.

446

ABBREVIATIONS USED

447

AMPK, AMP-activated protein kinase; ACC, Acetyl CoA Carboxylase; CD36,

448

cluster of differentiation 36; CPT1, carnitine palmityltransferase 1; FGF21,

449

fibroblast growth factor 21; GK, glucokinase; GLUT1, glucose transporter 1;

450

GLUT2, glucose transporter 2; GLUT4, glucose transporter 4; GS, glycogen

451

synthase; HDL-C, high-density lipoprotein cholesterol; NAFLD, non-alcoholic

452

fatty liver disease; PA, palmitic acid; PEPCK, phosphoenolpyruvate

453

carboxykinase; p-AMPK, phospho-AMP- activated protein kinase; p-ACC,

454

phospho-Acetyl-CoA Carboxylase; p-GS, phospho-glycogen synthase; SIRT1,

455

sirtuin1; TC, total cholesterol; TG, triglyceride

456

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Figure captions

615

Figure 1. Effects of PA and capsiate on HepG2 cell viability. (A) HepG2 cells

616

were treated with different concentrations of PA (0, 0.125, 0.25, 0.5 and 1.0

617

mM) for 48 h. (B) HepG2 cells were treated with different concentrations of

618

capsiate (0, 12.5, 25, 50, 100, 200 and 400 μM) for 24 h. (C) HepG2 cells were

619

treated with 0.25 mM PA for 24h and then co-incubated with different

620

concentrations of capsiate (0, 12.5, 25, 50, 100, 200 and 400 μM) for an

621

additional 24 h. Data are presented as mean ± SD and analyzed with

622

one-way ANOVA (n = 3). (*) P < 0.05 versus the control group.

623

Figure 2. Effects of capsiate on lipid accumulation in PA-treated HepG2 cells.

624

All groups were stained with Oil red O to observe lipid droplets at 400×

625

magnification. Scale bar=50 μm. (A) HepG2 cells were cultured in the absence 26

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of PA and capsiate; (B) HepG2 cells were treated with 0.25 mM PA for 48 h; (C

627

- E) HepG2 cells were treated with 0.25 mM PA for 24 h, then the cells which

628

exposed to PA were treated with (C) 25 μM capsiate, (D) 50 μM capsiate or (E)

629

100 μM capsiate for 24 h.

630

Figure 3. Effects of capsiate on TG, TC, HDL-C and glycogen contents in

631

PA-treated HepG2 cells. HepG2 cells were treated with or without 0.25 mM PA

632

for 24 h. Then the cells which exposed to PA were treated with capsiate (0, 25,

633

50, and 100 μM) for 24 h. (A) TG, (B) TC, (C) HDL-C levels of each group were

634

assayed and expressed in the bar chart. (D) Glycogen content of each group

635

was assayed and expressed in the bar chart. Data are presented as mean ±

636

SD and analyzed with one-way ANOVA (n = 3). (a) P < 0.05 versus the control

637

group; (b) P < 0.05 versus the PA-alone group.

638

Figure 4. Effects of capsiate on the expressions of lipid metabolism-related

639

proteins in PA-treated HepG2 cells. HepG2 cells were treated with or without

640

0.25 mM PA for 24 h. Then the cells which exposed to PA were treated with

641

capsiate (0, 25, 50, and 100 μM) for 24 h. (A) Protein expression of CD36, (B)

642

phosphorylation of ACC and (C) expression of CPT1A were quantified by

643

densitometry, and the relative intensities are expressed in the bar chart. Data

644

are presented as mean ± SD and analyzed with one-way ANOVA (n = 3). (a)

645

P < 0.05 versus the control group; (b) P < 0.05 versus the PA-alone group.

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646

Figure 5. Effects of capsiate on the expressions of glucose

647

metabolism-related proteins in PA-treated HepG2 cells. HepG2 cells were

648

treated with or without 0.25 mM PA for 24 h. Then the cells which exposed to

649

PA were treated with capsiate (0, 25, 50, and 100 μM) for 24 h. Protein

650

expressions of (A) GLUT1, (B) GLUT2, (C) GLUT4, (D) GK, (E) PEPCK, (F)

651

GS and phosphorylation of GS were quantified by densitometry, and the

652

relative intensities are expressed in the bar chart. Data are presented as mean

653

± SD and analyzed with one-way ANOVA (n = 3). (a) P < 0.05 versus the

654

control group; (b) P < 0.05 versus the PA-alone group.

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Figure 6. Effects of capsiate on the expressions of key metabolic regulators in

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HepG2 cells. (A, B, E) HepG2 cells were treated with or without 0.25 mM PA

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for 24 h, then the cells which exposed to PA were treated with capsiate (0, 25,

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50, and 100 μM) for 24 h. (A) Protein expression of AMPKα, phosphorylation of

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AMPKα, expressions of (B) SIRT1 and (E) FGF21 were quantified by

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densitometry, and the relative intensities are expressed in the bar chart. Data

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are presented as mean ± SD and analyzed with one-way ANOVA. (a) P