Flavonoid Glycosides from Fenugreek Seeds Regulate Glycolipid

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Biotechnology and Biological Transformations

Flavonoid Glycosides from Fenugreek Seeds Regulates Glycolipid Metabolism by Improving Mitochondrial Function in 3T3-L1 Adipocytes in Vitro G L, Yuwei Wang, Zhenhua Wang, Wenna Zhou, Na Hu, Gang Li, and Honglun Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00179 • Publication Date (Web): 10 Mar 2018 Downloaded from http://pubs.acs.org on March 15, 2018

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Journal of Agricultural and Food Chemistry

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Flavonoid Glycosides from Fenugreek Seeds Regulates Glycolipid

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Metabolism by Improving Mitochondrial Function in 3T3-L1

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Adipocytes in Vitro

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Guangxiang Luana,c,d, Yuwei Wanga,c,d, Zhenhua Wangb, Wenna Zhoua,c,d, Na Hua,c,

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Gang Lib*, Honglun Wanga,c*

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a

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Biology, Chinese Academy of Sciences, Xining 810008, P. R. China

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b

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University, Yantai 264005, P. R. China

Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau

Center for Mitochondria and Healthy Aging, college of Life Sciences, Yantai

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c

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d

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*Address correspondence to these two authors:

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23 Xin'ning Road, Xining, Qinghai, 810008 P.R. China (Honglun Wang: E-mail:

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[email protected]; Telephone: +8613997384106; Fax: +869716143857); 30

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Qingquan Road, Yantai, 264005 P.R. China (Gang Li: E-mail: [email protected];

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Telephone: +8613678659123; Fax: +865356902638)

Key Laboratory of Tibetan Medicine Research of Qinghai Province University of Chinese Academy of Sciences, Beijing 100049, P. R. China

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ACS Paragon Plus Environment


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Abstract: Fenugreek is a well-known annual herb widely used in both medicine and

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food. Four flavonoid glycosides have been separated from fenugreek seeds in our

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previous study. In this study, the effects of the four flavonoid glycosides on regulating

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glycolipid metabolism and improving mitochondrial function were investigated.

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Isoorientin showed a very significant activity among these flavonoid glycosides.

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Firstly, isoorientin decreased the accumulation of lipid droplets in 3T3-L1

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preadipocytes by reducing the expression of adipokines including PPARγ, C/EBPα

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and FAS. Secondly, isoorientin restored insulin-stimulated glucose uptake in

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dexamethasone-induced insulin resistant 3T3-L1 adipocytes by reactivating Akt and

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AMPK. Finally, isoorientin improved mitochondrial dysfunction induced by

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dexamethasone

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dexamethasone-induced decrease of mitochondrial membrane potential (MMP) and

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intracellular ATP production, reduced accumulation of intracellular reactive oxygen

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species (ROS) and protected mitochondrial DNA (mtDNA) from oxidative damage.

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At the same time, mitochondrial biogenesis is promoted. Therefore, isoorientin may

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be an attractive candidate as a glucose-lowering and insulin resistance improving

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agent for the treatment of diabetes.

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Keywords: Fenugreek seeds, Flavonoid glycosides, Isoorientin, Insulin resistance,

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3T3-L1 adipocytes, Mitochondrial function.

in

3T3-L1

adipocytes.

Isoorientin

2


also

reversed

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Introduction

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Obesity is a major risk factor for metabolic and cardiovascular diseases, and

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therefore is a risk to public health1. The obese condition is generally accompanied by

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excess accumulation of lipids and triglycerides (TG) synthesized by fatty acid

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synthase (FAS) in adipose tissue2-4. Adipokines, such as peroxisome proliferator

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activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBPα), act in

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tandem to regulate adipocyte differentiation5. Obesity-related insulin resistance is

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characterized by reduced glucose uptake in target tissues or cells due to impaired

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insulin signaling transduction and subsequent diminished glucose transport. In

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adipocytes, inactivation of insulin signaling molecules reduces the translocation of

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intracellular glucose transporter 4 (GLUT4) to the plasma membrane and

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consequently impairs glucose import into the cells 6, 7.

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Mitochondria can be recognized as a heart of the cell which modulates various

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cellular homeostasis such as cell proliferation, calcium homeostasis, detoxification of

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ammonia (in the liver), programmed cell death (apoptosis), oxidative stress (a

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hallmark of ageing process) and other related metabolic processes8. Studies recently

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have demonstrated an association between adipocyte mitochondrial dysfunction,

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obesity and insulin resistance9-11. Wilson-Fritch et al reported that there is a clear link

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between insulin resistance and reduced mitochondrial function12. Similar results have

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also been obtained in the insulin resistant 3T3-L1 adipocyte model by our previous

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experimental results.

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Fenugreek (Trigonella foenum-graecum L.), a well-known annual plant 3



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belonging to legume family, widely distributed in China, India, and North African

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countries and extensively used in both medicine and food13. The whole grass, flowers

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and seeds of fenugreek are used in Traditional Chinese medicine for the treatment of

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hypertension, hyperlipidemia and immune diseases14-17. Recent researches also proved

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that fenugreek can lower the blood glucose levels and improve lipid metabolism18-20,

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but the real effective components are unclear yet. Fenugreek has been reported to

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contain galactomannans, saponins, alkaloids, flavonoids, polyphenol stilbenes and

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amino acids21.

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isovitexin have been separated by high-speed counter-current chromatography

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(HSCCC) from fenugreek seeds for the first time in our previous study22.

Four flavonoid glycosides including orientin, isoorientin, vitexin and

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In our previous study, the hypoglycemic effects of extracts from fenugreek on

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STZ-induced type 2 diabetic mice fed with a high-fat diet were also proved. In the

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current study, we investigated the effects of flavonoid glycosides (Figure 1) from

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fenugreek seeds on glucose uptake and lipid accumulation in 3T3-L1 adipocytes. The

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mechanism was also discussed in terms of Akt and APMK phosphorylation pathway

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and mitochondrial dysfunction.

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Materials and Methods

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3T3-L1 Preadipocytes Differentiation and Induction of Insulin Resistance

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3T3-L1 cells were purchased from the cell bank of the Institute of Biochemistry

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and Cell Biology of Shanghai (shanghai, China) and grown in high glucose (4.5 g/L

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of glucose) Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, USA)

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containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C in a 4


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humidified incubator at 5% CO2. After 2 d of cells confluency, differentiation was

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induced by incubation in a similar medium supplemented with 0.5 mM

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3-isobuty-1-methylxanthine (IBMX; Sigma, St. Louis, MO, USA), 1 µM

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dexamethasone (Sigma, St. Louis, MO, USA) and 10 µg/mL insulin (Sigma, St. Louis,

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MO, USA) for 2 d (days 0-2) and then in a medium containing 10 µg/mL insulin for

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another 2 d (days 2-4). On the 4th day, the medium was replaced with DMEM

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containing 10% FBS, which was then replaced with the same medium every 2 d

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thereafter. Differentiated cells were used when at least 95% of the cells exhibited an

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adipocyte phenotype i.e. accumulation of lipid droplets. To investigate the effect of

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fenugreek flavonoid glycosides on adipocyte differentiation, four flavonoid

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glycosides (10 µM) were added together with the differentiation medium on days 0-4,

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respectively.

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An insulin-resistance adipocyte model was established as previously described

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with minor modifications23. On the 8th day after induction, the fully differentiated

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3T3-L1 adipocytes were treated with 1 µM dexamethasone for 48h. Cultured 3T3-L1

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adipocytes exposed to dexamethasone become insulin resistant as assessed by the

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ability

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2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) uptake

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and Akt phosphorylation level. To investigate the effect of fenugreek flavonoid

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glycosides on insulin resistance and mitochondrial dysfunction, 10 µM flavonoid

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glycosides were added together with dexamethasone.

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Cell Viability Assay

of

insulin-stimulated

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Preadipocytes in 150 µL growth medium were seeded in 96-well plates at 5 × 103

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cells per well in DMEM containing 10% FBS and incubated for 24 h until confluency.

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Cells were then treated with 0-100 µM flavonoid glycosides for 48 h. The MTT

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solution (5 mg/mL) was added to each well and the cells were incubated for 4 h in the

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incubator. The resultant formazan product was dissolved by the addition of 100 µL of

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DMSO. Absorbance was detected at 490 nm using a Multi-Mode Detection Platform

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(Molecular Devices, USA) 24.

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Oil-Red O Staining

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After differentiation on Lab-Tek® chambered cover glasses (Nalge Nunc

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International, Naperville, IL, USA) in 24-well plates, cells were washed twice with

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PBS and fixed with 4.0% formaldehyde for 30 min at room temperature. After

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washing with PBS three times, the cells were stained with 3.6 mg/mL Oil-Red O

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dye/60% isopropanol solution for 30 min. Excess Oil-Red O dye was washed out

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three times with 70% ethanol24. Stained oil droplets in 3T3-L1 cells were

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photographed by light microscopy (Olympus, Japan).

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TG Synthesis in Adipocyte Differentiation

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After differentiation in 12-well plates, cells were washed with ice-cold PBS

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twice. The cells were then repeatedly frozen and thawed at -80/37°C until the cells

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were fully disrupted. After addition of 100 µL PBS, cells were removed from the plate

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using a cell scraper and the mixed liquid was collected for assessment of TG

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concentration using the kit (Nanjing Jiancheng Bioengineering Institute, Nanjing,

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China). Intracellular TG content was normalized to the concentration of total protein

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measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc., 6


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Rockford, IL, USA)7.

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Flow Cytometric Analysis of 2-NBDG Uptake

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3T3-L1 preadipocytes were gently seeded into 6-well plates (1.5×105 cells/well).

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At the end of insulin resistant induction, cells were stimulated with or without 100

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nmol/L insulin for 15 min and then all culture medium was removed from each well

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and replaced with glucose free culture medium containing 100 µM 2-NBDG and

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incubated for another 30 min. The fluorescence was measured (excitation at 485/20

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nm and emission at 540/20 nm) using a FACS AriaTM Flow Cytometer (Becton

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Dickinson, San Jose, CA, USA). The data presented are the mean fluorescent signals

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for 20,000 cells.

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Western Blot Analysis

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Cells in 6-well plates were washed twice with ice-cold PBS and harvested in 100

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µL lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40 and

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0.1% SDS. Cell lysates were centrifuged at 12,000 rpm for 20 min at 4 °C25. The

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protein content of the supernatant was measured using the BCA assay reagent kit. Cell

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lysates were separated by SDS-PAGE gel electrophoresis and transferred onto a

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PVDF membrane using a Bio-RAD electrophoresis equipment. The membrane was

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blocked with 5% skim milk for 1h at room temperature and hybridized with primary

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antibodies (CST, USA) overnight at 4 °C. After incubation with horseradish

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peroxidase conjugated secondary antibody (ABGENT, Suzhou, P. R. China) for 1h at

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room temperature, immunoreactive proteins were detected using a Super Signal West

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Pico Chemiluminescent Substrate Kit according to the manufacturer's instructions25. 7



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Western blot bands were visualized using a 5200 Multi Luminescent Image Analyzer

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(Tanon Science & Technology Co., Ltd. Shanghai, China).

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Gene Expression Assay

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Gene expression was detected by reverse transcription real-time quantitative

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PCR (RT-qPCR). Total RNA was extracted using trizol reagent (Invitrogen, Carlsbad,

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CA, USA) and quantified by spectrophotometry at 260 nm. According to the

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manufacturer’s instructions, cDNA was synthesized from 1 µg of total RNA by using

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an M-MLV reverse transcriptase kit (Promega A3500; Promega, Madison, WI, USA).

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Real-time quantitative PCR (qPCR) was performed using the SYBR Premix Ex Taq II

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(Takara Bio INC) on a Rotor-Gene Q Sequence Detection System (QIAGEN,

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Germany)26. β-actin was used as the reference in the comparative CT method27 (2-△△Ct)

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to determine the relative changes in the target samples. The sequences of the primers

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for each gene are shown in Table 1.

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ATP Content Measurement

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ATP content was measured using a luciferase-based luminescence enhanced

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ATP assay kit (Beyotime, Shanghai, China). According to the manufacturer’s

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instructions, cells were washed twice with ice-cold PBS and homogenized in an

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ice-cold ATP releasing buffer. ATP concentrations were then determined using an

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ATP standard with a SpectraMax Paradigm Multi-Mode Microplate Reader

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(Molecular Devices, CA, USA).

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MMP Detection

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JC-1 (Sigma, St. Louis, MO, USA) was used to determine mitochondrial 8


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membrane potential (MMP). After treatment, cells were incubated with 10 µM JC-1

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in the dark under 37 ℃ for 30 min and then washed twice with pre-warmed PBS. The

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cells were suspended in pre-warmed PBS and analyzed using a FACS AriaTM Flow

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Cytometer (Becton Dickinson, San Jose, CA, USA) with 488 nm excitation and green

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or orange-red emission wavelengths28.

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Determination of Intracellular ROS Generation

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2’, 7’-dichlorofluorescein diacetate (DCFH-DA; Sigma, St. Louis, MO, USA)

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was used to determine the production of intracellular reactive oxygen species (ROS)

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with confocal laser microscopy and flow cytometry, simultaneously. For confocal

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laser microscopy, cells were cultured on cover slips in 24-well plates. After treatment,

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cells were incubated with 10 µM DCFH-DA in the dark under 37 ℃ for 30 min,

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washed twice with pre-warmed PBS, and then visualized using a confocal laser

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scanning microscope (OLYMPUS, Japan) with 488 nm excitation and 525 nm

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emission wavelengths. For flow cytometry, cells were seeded in 6-well plates. After

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treatment, cells were incubated with 10 µM DCFH-DA in the dark under 37 ℃ for

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30 min and then washed twice with pre-warmed PBS. The cells were suspended in

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pre-warmed PBS and analyzed using a FACS AriaTM Flow Cytometer (Becton

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Dickinson, San Jose, CA, USA) with 488 nm excitation and 525 nm emission

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wavelengths28.

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Detection of mtDNA Copy Number

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Relative amounts of mitochondrial DNA (mtDNA) were determined by qPCR as

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previously described using SYBR Premix Ex Taq II (Takara Bio INC) on a 9



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Rotor-Gene Q Sequence Detection System (QIAGEN, Germany). Briefly, DNA was

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isolated from adipocytes using DNA extraction kit (TIANGEN, Beijing, China) and

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quantified by spectrophotometry at 260 nm. Two primer sets were used for PCR

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analysis. Amplification of a 117 nucleotide long mtDNA fragment within mtDNA

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was used for the quantification of mtDNA26. The nuclear gene β-actin was used to

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normalize the results. The ratio of mtDNA to nuclear DNA (nDNA) reflects the

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concentration of mitochondria per cell27. The sequences of the primers are shown in

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Table 1.

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Detection of mtDNA Damage by Long PCR

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DNA was isolated from adipocytes using a DNA extraction kit (TIANGEN,

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Beijing, China) and then quantified by spectrophotometry at 260 nm and qualified by

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0.7% agarose gel electrophoresis, respectively. Long PCR experiments allowed the

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detection of mtDNA lesions that hamper the progression of polymerases and altering

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replication29,30. The long PCR technique is based on the amplification of a long

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(8636-bp) and a short (117-bp) mtDNA fragment and the selected primers are listed in

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Table 1. PCR reactions were performed with the Veriti 96 Well thermal Cycler long

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PCR system (Applied Biosystems, Foster City, CA, USA) as recommended by the

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manufacturer. The long fragment reaction thermocycler profile included initial

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denaturation at 94°C for 2 min, 40 cycles of 94°C for 45 s, 61°C for 10 s, 68°C for 8

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min and a final extension at 68°C for 7 min using Long Amp Taq 2x Master Mix

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(NEB, Ipswitch, MA, USA). Short fragment reaction thermocycler profile included

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initial denaturation at 94°C for 10 min, 40 cycles of 94°C for 10 s, 65°C for 15 s, 10


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72°C for 20 s and a final extension at 72°C for 10 min using Taq DNA Polymerase

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(Thermo Scientific™, USA). PCR products were subjected to electrophoresis on

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ethidium bromide-containing agarose gels and the intensity of each PCR fragment

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was determined using a transilluminator (Tanon Science & Technology Co.,

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Ltd. Shanghai, China). The long/short mtDNA intensity ratio was thus calculated for

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each sample29.

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Statistical Analysis

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Data were shown as means ± S.D. from three independent experiments.

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Statistical analysis was performed by one way ANOVA or student’s t-test using the

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statistical analysis software SPSS 18.0. P < 0.05 was considered statistically

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significant.

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Results

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Fenugreek Flavonoid Glycosides Inhibited 3T3-L1 Preadipocytes Differentiation

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and Lipogenesis

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In order to determine the safe concentration range, the cytotoxicity of fenugreek

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flavonoid glycosides was measured by MTT assay. As shown in Figure 2a, four

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fenugreek flavonoid glycosides have no obvious effects on the proliferation of

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3T3-L1 preadipocytes at 0.1-100 µM concentration for 48 h. During the

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differentiation of 3T3-L1 preadipocytes, lipid droplets formation is a typical

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phenomenon and used as a marker of differentiation5. The inhibitory effects of

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fenugreek flavonoid glycosides on TG contents and oil droplet accumulation in

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differentiated adipocytes were analyzed. TG content of fully differentiated adipocytes

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was quantified by directly measuring TG levels and oil droplet accumulation in cells

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with Oil-Red O stain. As shown in Figure 2b and c, more than 95% differentiated

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cells were filled with bigger oil droplets and more high level of TG content were

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observed in fully differentiated 3T3-L1 cells, whereas rare was found in

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undifferentiated control. However, the cells treated with isoorientin and vitexin had

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remarkably less oil droplet accumulation and lower level of TG contents (P < 0.01).

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Fenugreek Flavonoid Glycosides Downregulated Adipogenic Genes Expression

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Adipocyte differentiation from 3T3-L1 preadipocytes is associated with the

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expression of adipocyte-specific genes such as PPARγ and C/EBPα5. We therefore

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investigated the expression of these proteins (Figure 2d). Our results demonstrated

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that the expression levels of PPARγ and C/EBPα was very low in 3T3-L1

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preadipocytes. However, on day 8 post induction, these genes were highly expressed

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in differentiated cells. Treatment with four fenugreek flavonoid glycosides

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significantly reduced the expression of PPARγ and C/EBPα compared to fully

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differentiated control adipocytes. Further investigations were carried out to determine

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the effect of fenugreek flavonoid glycosides on the regulation of the adipogenic target

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gene FAS, which plays the role of synthetic fatty acids31. As shown in Figure 2d, all

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of the four fenugreek flavonoid glycosides dramatically decreased the expression

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level of FAS.

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Fenugreek Flavonoid Glycosides Restored Glucose Uptake by Activating Akt and

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AMPK

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To investigate the effects of fenugreek flavonoid glycosides on glucose uptake in

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the insulin resistant 3T3-L1 adipocytes, fluorescent deoxyglucose analog (2-NBDG)

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was used to measure the rates of glucose uptake. 2-NBDG has been wildly used in

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various studies, especially for exploring cellular metabolic functions associated with 12


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GLUT systems32. As shown in Figure 3a, 3T3-L1 adipocytes in the presence of 1 µM

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dexamethasone take up much less 2-NBDG than cells in the absence of

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dexamethasone (p

Flavonoid Glycosides from Fenugreek Seeds Regulate Glycolipid

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