Capsaicin ameliorates the redox imbalance and glucose metabolism

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Bioactive Constituents, Metabolites, and Functions

Capsaicin ameliorates the redox imbalance and glucose metabolism disorder in insulin-resistance model via circadian clock-related mechanisms MUWEN LU, Yaqi Lan, Jie Xiao, Mingyue Song, Chengyu Chen, Caowen Liang, Qingrong Huang, Yong Cao, and Chi-Tang Ho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04016 • Publication Date (Web): 18 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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Journal of Agricultural and Food Chemistry

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Capsaicin ameliorates the redox imbalance and glucose metabolism disorder in

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insulin-resistance model via circadian clock-related mechanisms

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Muwen Lu†, Yaqi Lan†, Jie Xiao†, Mingyue Song†, Chengyu Chen‡, Caowen Liang†,

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Qingrong Huang§, Yong Cao†,* and Chi-Tang Ho§,*

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†

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Food Science, South China Agricultural University, Guangzhou 510642, China

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‡

Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of

College of Natural Resources and Environment, South China Agricultural University,

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Guangzhou 510642, China.

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§

Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA

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* To whom correspondence should be addressed. Tel: 848-932-5553 (CH). Fax: 732-932-6776;

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Email: [email protected] (CH); [email protected] (YC)

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ACS Paragon Plus Environment


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ABSTRACT

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Circadian rhythms are closely associated with metabolic homeostasis. Metabolic

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disorders can be alleviated by many bioactive components through the controlling of clock

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gene expressions. Capsaicin has been demonstrated with many beneficial effects including

21

anti-obesity and anti-insulin resistance activities, yet whether the rhythmic expression of

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circadian clock genes are involved in the regulation of redox imbalance and glucose

23

metabolism disorder by capsaicin remains unclear. In this work, the insulin resistance was

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induced in HepG2 cells by the treatment of glucosamine. Glucose uptake level, reactive oxygen

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species (ROS), H2O2 production and mitochondrial membrane potential (MMP) were

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measured with/without capsaicin co-treatment. The mRNA and protein expressions of core

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circadian clock genes were evaluated by RT-qPCR and western blot analysis. Our study

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revealed that circadian misalignment could be ameliorated by capsaicin. The glucosamine-

29

induced cellular redox imbalance and glucose metabolism disorder were ameliorated by

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capsaicin in a Bmal1-dependent manner.

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KEYWORDS: Capsaicin; circadian clock, insulin resistance, redox homeostasis

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INTRODUCTION

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Circadian rhythms are biological variables that oscillate cyclically with a period close to

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24 h, allowing the organism to make adaptations to the constantly changing environment,

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including light, temperature and nutrients.1 The central circadian clock is located in the

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hypothalamus suprachiasmatic nucleus (SCN), which is composed of multiple circadian

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oscillators operated by two interlocking transcription/translation feedback loop (TTFL).2 The

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24-h rhythmic circadian gene expression regulated by TTFL is driven by four integral clock

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proteins: two activators (CLOCK and BMAL1) and two inhibitors (PER and CRY), as well as

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by other kinases and phosphatases.3 CLOCK and BMAL1 form CLOCK/BMAL1 heterodimer

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and bind to the promoters of clock-controlled genes at E-boxes (5’-CACGTG-3’) in the nucleus,

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inducing the transcription of other circadian genes such as Pers and Crys. As the protein

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concentration of the PER and CRY accumulates, the polymers will be formed to inhibit the

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transcription mediated by CLOCK/BMAL1 heterodimer.4

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Metabolic disorders have become serious global health issues negatively affecting lives

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of many people, which occur when the body's usual metabolic processes are disrupted.5,6

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Insulin resistance, as a hallmark of the metabolic syndrome, occurs when cells are unable to

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respond normally to the hormone insulin.7 Insulin resistance is intimately linked to a variety of

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metabolic syndromes, such as hypertension, hyperlipidemia and atherosclerosis.8

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Growing evidence indicates that circadian rhythm is closely related with metabolic

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homeostasis and the disruption of circadian rhythms results in metabolic disorders.6,9 Therefore,

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many metabolic systems may in turn affect the function of clock genes and circadian systems.

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Various studies revealed that dietary bioactive component could regulate metabolic disorders

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via the involvement of circadian clock genes.10-12 Qi, et al. reported that tea polyphenols could

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alleviate metabolic syndrome and mitochondrial dysregulation in a circadian gene-dependent

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manner.13 According to Sun, et al., resveratrol could attenuate the high-fat diet (HFD)-induced

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circadian misalignment of lipid metabolism in male C57BL/6 mice.14 Cichoric acid, a

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hydroxycinnamic acid occurs in a variety of plant species, could prevent free-fatty-acid-

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induced disorders of lipid metabolism through the modulation of the circadian gene Bmal1

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expressions in hepatocytes.15

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Capsaicin, the main capsaicinoid in chili peppers with pungent and spicy flavor,16-18 has

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many

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cardioprotective,25 anti-oxidation26 and anti-obesity activities.27,28 Kang, et al. reported the

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administration of capsaicin could reduce obesity-related metabolic disorders such as insulin

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resistance and hepatic steatosis induced by HFD in male C57BL/6 mice.29 Later, they found

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that capsaicin attenuated the metabolic dysregulation by enhancing expressions of adiponectin

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and its receptor in obese diabetic KKAy mice that exhibits serious insulin resistance.30 Jeong,

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et al. treated Sprague Dawley (SD) rats with capsaicin water suspension and revealed that

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capsaicin could regulate the expression of circadian clock gene Per2.31 Therefore, capsaicin

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may exert the preventative effect on insulin resistance by regulating the circadian clock gene

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expressions. However, it remains unclear whether the rhythmic expressions of circadian clock

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genes are involved in glucosamine-induced insulin resistance ameliorated by capsaicin in

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human hepatocytes.

beneficial

effects

such

as

anti-inflammation,19

anti-cancer,20-23

analgesic,24

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In this work, the protective effect of capsaicin on circadian disruption triggered by

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glucosamine was investigated using HepG2 cell line. The glucosamine-induced oxidative

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stress and mitochondrial dysfunction relieved by capsaicin were also evaluated in respect of

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the core circadian clock gene expressions.

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MATERIALS AND METHODS

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Materials

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Capsaicin (purity ~99%) was purchased from Ji’an Shengda Fragrance Oils Company

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(Ji’an, Jiangxi, China). Milli-Q water (18.3 MΩ) was used in all experiments.

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Cell Culture and Viability Assay

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HepG2 cell line was obtained from Collection of Cell Cultures of the Fourth Military

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Medical University of the People's Liberation Army (Xi’an, Shaanxi, China). Modified

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Roswell Park Memorial Institute (RPMI)-1640 medium was purchased from Thermo Fisher

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Scientific (Waltham, MA, USA). HepG2 were cultured in RPMI medium supplemented with

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10% fetal bovine serum (FBS), penicillin (100 kU/L) and streptomycin (100 mg/L). Cells were

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maintained at 37 °C in a humidified atmosphere of 5% CO2.

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The MTT assay was used to evaluate the viability of HepG2 cells. Cells were seeded at a

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density of 2.0 × 104 cells/well in 96-well plates. After an overnight incubation, glucosamine

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(20 mM) and capsaicin with different concentrations were added. At the end of each treatment,

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MTT solution (Sigma, St. Louis, MO, USA) was added and incubated at 37 °C for 4 h.

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Formazan crystal which displayed a purple color was detected by a by Bio-Rad iMark

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microplate reader at 490 nm (Bio-Rad, Hercules, CA, USA). All experiments were performed

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in triplicate.

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Glucose Uptake

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The glucose uptake study was performed according to the reported method with slight

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modifications.32 HepG2 cells were treated with 20 mM glucosamine to induce insulin

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resistance, and then co-treated with 50 μM capsaicin for 18 h after incubating with 100 nM

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insulin. The supernatant was measured at wavelength of 540 nm by a glucose assay kit (Nanjing

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Jiancheng Bioengineering Institute, Nanjing, China). The optical density of each sample was

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measured and the glucose concentration was calculated. Six replicate wells were established

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and all experiments were performed in triplicate.

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Measurement of Reactive Oxygen Species (ROS) and H2O2 Level

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Intracellular ROS levels were measured by using ROS assay kit (Beyotime Biotechnology,

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Nanjing, China), which could be used to measure the hydroxyl, peroxyl, and other reactive

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oxygen species activity within a cell. The cells were treated with 2´,7´-dichlorofluorescein-

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diacetate (DCFDA), which could be converted to the fluorescent dichlorofluorescein (DCF) by

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intracellular ROS. Fluorescence was read using an excitation wavelength at 495 nm and

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emission wavelength at 529 nm in the plate reader Synergy H1 (BioTek, Winooski, VT, USA).

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The extracellular H2O2 levels were detected by the Amplex Red hydrogen

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peroxide/peroxidase assay kit (Invitrogen, Carlsbad, CA, USA). The Amplex Red reagent, in

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combination with horseradish peroxidase (HRP), could react with H2O2 and produce a highly

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fluorescent resorufin. Fluorescence intensity was measured by microplate reader using an

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excitation wavelength at 490 nm and emission wavelength at 535 nm.

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Measurement of Mitochondrial Membrane Potential (MMP)

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MMP assay kit with JC-1 (Beyotime, Nanjing, China) was applied to measure the

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mitochondrial membrane potential (MMP). After incubation overnight, cells were centrifuged,

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resuspended in phosphate-buffered saline (PBS) and stained in cationic dye JC-1. Fluorescence

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were detected by Synergy Neo2 hybrid multi-mode microplate reader (BioTek, Winooski, VT,

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USA). The MMP levels was indicated by the intensity ratio of green/red fluorescence.

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RNA Extraction and Real-time PCR

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Total RNA was extracted from HepG2 cells by a RNA isolator (TaKaRa, Dalian, China),

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and cDNA was synthesized using the Primescript RT reagent (TaKaRa). The relative mRNA

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quantification was analyzed by RT-qPCR using a SYBR green I dye (TaKaRa) and the CFX96

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Touch real-time PCR detection system (Bio-Rad). Gene-specific mouse primers used in this

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study were summarized in Table 1. The relative transcript level of each target gene was

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calculated according to the 2−Ct method for gene normalization to GAPDH.33

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Western Blot Analysis

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Cell lysates were prepared by solubilizing in SDS sample buffer. After proteins were

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transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt,

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Germany), they were stained for visualization and identified by immunodetection. Antibodies

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including Anti-CLOCK (ab93804), Anti-BMAL1 (ab93806), Anti-CRY1 (ab54649), Anti-

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CRY2 (ab155255), Anti-PER1 (ab136451) Anti-PER2 (ab179813) and Anti-GAPDH

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(ab181602) were purchased from Abcam (Abcam, Cambridge, MA, USA). Quantitative

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analysis of western blot was achieved by the Quantity One v4.6.2 (Bio-Rad).

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Statistical Analysis

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All of the data are expressed as means ± standard error. Variances between groups were

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determined using one-way ANOVA by SPSS software. Significance level at p < 0.05, 0.01 ,

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and 0.001 were considered statistically significant.

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RESULTS AND DISCUSSION

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Capsaicin Alleviated Glucosamine-impaired Glucose Uptake in HepG2 Cells

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Different concentrations of capsaicin (0, 25, 50, 75, 100 μM, respectively) were applied

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to HepG2 cells to examine the effects of capsaicin on cell viability during proliferation.

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According to Figure 1A, the concentration of 50 μM was selected with 84.67% cell viability.

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Studies revealed that insulin resistance could be induced by glucosamine through the

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hexosamine biosynthesis pathway, causing insulin disturbances and glucose intolerance.34 In

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this work, HepG2 cells were treated with glucosamine at concentration of 20 mM to induce

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insulin resistance (Figure 1B).

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Glucose uptake in cells treated with capsaicin and glucosamine (20 mM) is presented in

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Figure 1C. The insulin-stimulated glucose uptake was reduced significantly from 100% to

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33.94% after glucosamine treatment (p < 0.001) in HepG2 cells. After the capsaicin treatment

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for 18 h, the glucose uptake increased effectively from 33.94% to 72.8% (p < 0.001), indicating

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that capsaicin could alleviate the glucosamine-impaired glucose uptake. The glucose uptake

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was further enhanced to 85.95% (p < 0.001) with the increase in capsaicin concentration,

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suggesting that the glucosamine-induced insulin resistance could be alleviated by capsaicin in

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a dose-dependent manner in HepG2 cells.

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Regulation of Circadian Misalignment by Capsaicin in HepG2 Cells

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Studies revealed that the disruption of circadian clock was closely related with insulin

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resistance and obesity.5 Mi, et al. and Zhu, et al. both reported that glucosamine treatment

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could induce insulin resistance in HepG2 cells,10,35 which resulted in the decrease in the

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transcription level of Clock and Bmal1. In this work, circadian misalignment was induced by

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the treatment of glucosamine in the insulin-resistance model. The expression levels of circadian

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rhythm genes Clock, Bmal1, Per1, Per2, Cry1, and Cry2 in HepG2 cells were measured by

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RT-qPCR, and results are presented in Figure 2 (A-F). Most of the oscillating genes in the

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control group were expressed in a rhythmic pattern, such as Clock, Bmal1, Per2, Cry1 and

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Cry2. However, after glucosamine treatment, the mRNA expressions of both activators (Clock

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and Bmal1) and inhibitors (Per1) displayed relatively shallow oscillations, which were

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reversed by capsaicin treatment effectively.

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To further demonstrate the regulation effect of capsaicin in alleviating the circadian

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misalignment triggered by glucosamine in insulin resistance models, the protein expressions

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of core clock components in these groups were measured through western blot analysis.

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According to Figure 3 (A-F), relative shallow oscillations were observed in the glucosamine-

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treated group for the protein expressions of BMAL1, CRY1 and CRY2. The oscillatory

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behavior was recovered by capsaicin co-treatment efficiently, which was in consistent with the

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mRNA expression levels of clock genes. Therefore, capsaicin was proved to regulate the

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glucosamine-induced circadian clock disruption at both RNA and protein level in insulin-

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resistant HepG2 cell model.

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Inhibitory Effects of Capsaicin on the Glucosamine-induced ROS Production and Mitochondria Dysfunction in HepG2 Cells

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The pathophysiology of insulin resistance is complex and still incompletely understood,

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yet it has been reported to be intricately linked to mitochondrial dysfunction and reactive

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oxygen species (ROS) imbalance.36,37 The importance of ROS signaling and oxidative stress in

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the development of insulin resistance has been implicated in many studies.38 An excessive

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amount of ROS could damage cellular lipids, proteins, or DNA, leading to the inhibition of

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signal transduction pathways and disruption of normal cellular functions. As shown in Figure

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4 (A), the redox status in HepG2 cells was measured using DCFDA. Relative ROS level was

190

calculated and presented in Figure 4 (B). In comparation with the control, the relative ROS

9



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level was increased from 100.00% to 326.98% for the glucosamine-treated group, showing that

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glucosamine could induce the production of ROS in HepG2 cells and that the imbalance of

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ROS may promote mitochondrial dysfunction. The ROS level was reduced to 141.10% after

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the co-treatment of 50 M capsaicin, indicating that capsaicin had an inhibition effect on the

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production of excess ROS caused by glucosamine.

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During normal cellular metabolism, mitochondrial electron transport could result in the

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formation of hydrogen peroxide (H2O2).39 It was reported that excess H2O2 emission could

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inhibit the activities of specific mitochondrial enzymes and overall mitochondrial respiration,

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leading to insulin resistance in both rodents and humans.40 According to Figure 4 (C), the H2O2

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content in glucosamine-treated HepG2 cells was increased from 98.76% to 173.21% compared

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to the control group, while it was reduced to 112.92% due to the existence of capsaicin,

202

indicating that capsaicin was effective in inhibiting the glucosamine-induced H2O2 emission.

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The mitochondrial membrane potential (MMP, ΔΨm) was measured using JC-1 dye and

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presented as the ratio of green/red using fluorescence microscopy, as was shown in Figure 4

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(D). In healthy cells, ΔΨm was relatively high and JC-1 could aggregate with deep red

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fluorescence. However, for unhealthy cells with low ΔΨm, JC-1 existed in a monomeric form

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and exhibited green florescence. Therefore, the lower ratio of green/red fluorescence reflected

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a higher polarization of the mitochondrial membrane. Compared with the control group, the

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ratio of green/red fluorescence for glucosamine group was increased from 94.35% to 276.01%,

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which was recovered to 105.44% by capsaicin co-treatment, demonstrating the alleviating

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effect of capsaicin on glucosamine-induced mitochondria dysfunction in HepG2 cells.

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Alleviation of Cellular Redox Imbalance by Capsaicin via Regulating the Circadian Clock Genes in mRNA Levels.

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Compelling evidence suggested that many metabolic disorders could be prevented by

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functional food ingredients via regulating circadian gene expressions in HepG2 cells.15,41 To

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verify if the mRNA level of circadian clock genes could affect the modulation effect of

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capsaicin on cellular redox homeostasis, small interfering RNA (siRNA) was used in HepG2

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cells to decrease Bmal1 abundance. As shown in Figure 4 (A-B), by exposing to si-Bmal1, the

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relative ROS level increased from 141.01% to 202.54%, indicating that the inhibition effect of

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capsaicin on ROS production was weakened by the knockdown of Bmal1 gene. Same trend

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could be observed for the H2O2 level and the mitochondrial membrane potential in HepG2 cells.

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According to Figure 4 (C), the H2O2 concentration in si-Bmal1 group was increased from

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112.92% to 143.61% compared with capsaicin group, showing a reduced effect in suppressing

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the glucosamine-induced H2O2 emission. The green/red florescence ratio for the Bmal1-

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knocked down group was also raised from 105.44% to 192.43%, reflecting a diminished

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modulation effect on MMP by capsaicin in the presence of si-Bmal1 (Figure 4 D). Therefore,

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the regulation effect of capsaicin on cellular redox homeostasis is dependent on the mRNA

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level of the circadian clock gene Bmal1.

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Capsaicin Ameliorated Glucose Metabolism Disorder in HepG2 Cells in a Bmal1Dependent Manner

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To study the role of circadian clock gene Bmal1 in the regulation of glucose metabolic

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disorder by capsaicin, Bmal1 was silenced using siRNAs and relative protein levels for core

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circadian clock genes were measured. Based on the western blot analysis shown in Figure 5(A-

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B), the protein level of BMAL1 was significantly reduced to 51.52% with si-RNA knockdown

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in HepG2 cells. Meanwhile, the relative protein expressions of CLOCK, CRY2, PER1 and

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PER2 were also suppressed compared with the control group, suggesting that Bmal1 as the

237

core clock gene could regulate the expression of other circadian genes involved in the

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transcriptional feedback loop, which was in consistent with previous studies.42-44 As shown in

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Figure 5(B), the treatment of glucosamine resulted in the down-regulation of protein levels of

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circadian genes, such as BMAL1, CLOCK, CRY1 and PER2, which was reversed by capsaicin

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co-treatment. However, the protein levels in capsaicin co-treated group failed to increase after

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Bmal1 deletion, demonstrating that the modulation effect of capsaicin on expression of

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circadian clock genes relied upon regular expression of Bmal1.

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According to Figure 5 (C), the glucose uptake was measured under different conditions.

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Compared with the control group, the glucose uptake in Bmal1- knockout group was reduced

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to 77.90%, suggesting that si-Bmal1 impaired the glucose metabolism in HepG2 cells. The

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glucosamine-induced glucose metabolic disorder was alleviated after capsaicin treatment with

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glucose uptake improved from 64.59% to 85.64%. The amelioration effect of capsaicin on

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glucose metabolic disorder was decreased after Bmal1 deletion with the glucose uptake level

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of 66.11%, showing that capsaicin mitigated glucose metabolic dysfunction in HepG2 cells in

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a Bmal1-dependent manner.

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In summary, our study revealed that capsaicin could alleviate the circadian misalignment

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and inhibit glucosamine-induced ROS production and mitochondria dysfunction in HepG2

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cells. The glucose metabolism disorder was also relieved by capsaicin through regulating the

255

rhythmic expression of circadian clock gene Bmal1. These findings could provide novel

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solutions in the prevention and treatment of obesity, insulin resistance as well as other

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metabolic disorders through the modulation of clock genes.

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Acknowledgements

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This work was financially supported by the National Natural Science Foundation of China (No.

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31901689).

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Conflict of interest

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The authors declare no competing financial interest.

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Abbreviations:

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BMAL1, brain and muscle arnt-like protein 1; CRY, cryptochrome. CAP, capsaicin; DCF,

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dichlorofluorescein; DCFDA, 2’,7’-dichlorofluorescein-diacetate; FL, fluorescence; GAPDH,

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glyceraldehyde 3-phosphate dehydrogenase; HFD, high-fat diet; HRP, horseradish peroxidase;

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H2O2, hydrogen peroxide; JC-1, tetraethyl benzimidazolyl carbocyanine iodide; MMP (ΔΨm),

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mitochondrial membrane potential; PER, Period circadian protein; ROS, reactive oxygen

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species; PVDF, polyvinylidene difluoride; SCN, suprachiasmatic nucleus; SD rat, Sprague

270

Dawley rat; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulphate-

271

polyacrylamide

272

transcription/translation feedback loop.

gel

electrophoresis;

siRNA,

small

273

13


interfering

RNA;

TTFL,


274

Table 1. Primer sequences used for quantitative real-time PCR analysis. Forward primer

Reverse primer

GAPDH TCAAGAAGGTGGTGAAGCAGG TCAAAGGTGGAGGAGTGGGT Bmal1

ATGGGGCTGGATGAAGACAA

CTGTTGCCCTCTGGTCTACA

Clock

ACGACGAGAACTTGGCATTG

GGTGTTGAGGAAGGGTCTGA

Per1

AAGTCCGTCTTCTGCCGTAT

TATCCGGGGAGCTTCGTAAC

Per2

AGCCGGAGTTAGAGATGGTG

TCTGCTCCTCCTTCTGTGTG

Cry1

GTCTACATCCTGGACCCCTG

CTGGGAAACACATCTGCTGG

Cry2

GGGAGGAGAGACAGAAGCTC

AATAGGGAGAGGGGAGGTGT

275 276

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Figure Captions

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Figure 1. Capsaicin alleviated glucosamine-impaired glucose uptake in HepG2 cells. (A)

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Relative viability of HepG2 cells treated with different concentrations of capsaicin measured

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by MTT assay; (B) Relative cell viability treated with 50 μM capsaicin and co-treated with

281

/without 20 mM glucosamine; (C) Glucose uptake in groups treated with capsaicin (25, 50 μM)

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and co-treated with/without glucosamine (20 mM). Data were presented as the mean value ±

283

SE (n≥ 6): (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P < 0.001 versus control group.

284

Figure 2. Capsaicin regulated glucosamine-induced circadian misalignment in HepG2 cells.

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(A-F) The mRNA expression levels of circadian rhythm genes Clock, Bmal1, Per1, Per2, Cry1,

286

and Cry2 in HepG2 cells measured by RT-qPCR and normalized to β-actin mRNA levels. Data

287

were presented as the mean value ± SE (n=3): (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P < 0.001

288

versus control group; (#) P < 0.05,( ##) P < 0.01 and (###) P < 0.001 versus glucosamine

289

group.

290

Figure 3. The effects of CAP on glucosamine-induced clock genes changes were determined

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by western blots. Clock, Bmal1, Cry1, Cry2, Per1 and Per2 were detected in HepG2 cells, and

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GAPDH was used as a loading control. (A)-(F) Densitometric analysis of the blots shown in

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G. Data were presented as the mean value ± SE (n=3): (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P

294

< 0.001 versus control group; (#) P < 0.05,( ##) P < 0.01 and (###) P < 0.001 versus

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glucosamine group.

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Figure 4. Capsaicin alleviated the imbalance in redox status induced by glucosamine in HepG2

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cells. HepG2 cells were cultured with/without glucosamine and co-treated with capsaicin for

298

18 h. (A)-(B) The cellular oxidation status in different groups detected using DCFDA. (C)

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Production of hydrogen peroxide (H2O2) measured by Amplex Red Hydrogen

300

Peroxide/Peroxidase Assay Kit. (D) The mitochondrial membrane potential reflected as the

15



301

ratio of green/red using fluorescence microscopy. Data were presented as the mean value ±

302

SE (n=3): (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P < 0.001 versus control group.

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Figure 5．Capsaicin ameliorated glucose metabolism disorder induced by glucosamine via

304

modulating the protein expression of circadian clock genes. HepG2 cells were transfected with

305

si-Bmal1 for 48 h, and then cultured with/without glucosamine and co-treated with capsaicin

306

for 18 h. β-actin were used as a loading control. (A) Representative western blots of core

307

circadian clock genes after treatment with glucosamine and capsaicin in HepG2 cells. (B)

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Densitometric analysis of the blots shown in A. (C) The glucose content in cells measured by

309

a glucose uptake analysis kit. Data were presented as the mean value ± SE (n= 3): (∗) P < 0.05,

310

(∗∗) P < 0.01, and (∗∗∗) P < 0.001 versus control group.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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