Black Soybean Leaf Extract Suppresses Hyperglycemia and Hepatic

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Bioactive Constituents, Metabolites, and Functions

Black Soybean Leaf Extract Suppresses Hyperglycemia and Hepatic Steatosis by Enhancing Adiponectin Receptor Signaling and AMPK Activation Hua Li, Un-Hee Kim, Jeong-Hyun Yoon, Hyeon-Seon Ji, Hye-Mi Park, Ho-Yong Park, and Tae-Sook Jeong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04527 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 13, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45 abstract Graphic

Journal of Agricultural and Food Chemistry

Blood

Liver

AdipoR1

AdipoR2

Adipose tissue

p AMPK IRS

p p

AKT

Insulin sensitivity

PPARα, PPARδ, PPARγ CPT-1, ACCs, FAS

TG

Adiponectin Insulin sensitivity

Lipid accumulation

Glucose GLUT-2

EBL QGs, IRGs Adiponectin TG Glucose

ACS Paragon Plus Environment


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Black Soybean Leaf Extract Suppresses Hyperglycemia and Hepatic Steatosis by

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Enhancing Adiponectin Receptor Signaling and AMPK Activation

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Hua Li, Un-Hee Kim, Jeong-Hyun Yoon, Hyeon-Seon Ji, Hye-Mi Park, Ho-Yong Park, Tae-

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Sook Jeong*

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Industrial Biomaterials Research Center, Korea Research Institute of Bioscience and

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Biotechnology, Daejeon 34141, Republic of Korea

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*Corresponding author at: Industrial Biomaterials Research Center, Korea Research Institute

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of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong, Daejeon 34141,

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Republic of Korea. Tel: +82-42-860-4558; fax: +82-42-861-2675.

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E-mail address: [email protected].

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1


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ABSTRACT

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Yellow soybean leaf extract including kaempferol glycosides and pheophorbides reduces

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obesity and plasma glucose levels. This study researched the molecular mechanisms underlying

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the glucose-lowering effect of the extract of black soybean leaves (EBL), which mainly

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contains quercetin glycosides and isorhamnetin glycosides, in high-fat diet (HFD)-induced

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obese diabetic mice and HepG2 cells. Twelve weeks of EBL supplementation decreased body

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weight, fasting glucose, glycated hemoglobin, insulin, triglyceride, and non-esterified fatty

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acids. Histological analyses manifested that EBL suppressed hepatic steatosis. Interestingly,

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EBL significantly improved plasma adiponectin levels and increased adiponectin receptor gene

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(AdipoR1 and AdipoR2) expression in the liver. EBL restored the effects of HFD on hepatic

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AMP-activated protein kinase (AMPK) and on family of peroxisome proliferator-activated

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receptors (PPARα, PPARδ, and PPARγ), which are associated with fatty acid metabolism and

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are downstream of AdipoRs. Hence, EBL effectively diminished hyperglycemia and hepatic

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steatosis through enhancing adiponectin-induced signaling and AMPK activation in the liver.

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KEYWORDS: Black soybean leaf; Hyperglycemia; Steatosis; Adiponectin; AMPK

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INTRODUCTION

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A number of diseases are closely interrelated to the development of type 2 diabetes

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including obesity, atherosclerosis, and fatty liver disease, which is caused by insufficient

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insulin production or insulin resistance. In addition to the pancreas, which releases insulin and

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glucagon to regulate blood glucose levels during feeding and fasting, the liver is the other key

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player in maintaining glucose homeostasis; it releases glucose during exercise, fasting, or

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pregnancy to satisfy the body’s increased needs. Hepatic steatosis increases the risk for the

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progression of hepatic insulin resistance in obese patients.1 Hepatic steatosis is characterized

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by accumulated lipid droplets in hepatocytes as vacuoles of triglyceride (TG) fat; it is a

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common pathogenic event in obese and diabetic mice and humans.2

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Adiponectin is an adipocytes produced hormone and its circulation levels are lower in

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obese diabetic patients.3 It decreases insulin resistance and exerts anti-apoptotic, antisteatotic,

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anti-inflammatory, and antilipogenic effects through activation of adiponectin receptor

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signaling.4 Full-length adiponectin is binding to adiponectin receptor-1 (AdipoR1) or AdipoR2

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and lowers the intracellular lipid content of hepatocytes by enhancing activity of AMP-

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activated protein kinase (AMPK) or peroxisome proliferator-activated receptor α (PPARα).

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Inhibition of gluconeogenesis by adiponectin lowers plasma insulin levels, which reduces

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insulin-mediated lipid accumulation and insulin-mediated inhibition of fatty acid utilization.

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Soybean [Glycine max (L.) Merr.], has become an increasingly important health food

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source because of its bioactive properties. Soybean seeds exist in various colors, including

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yellow, brown, green, and black seed coats. The leaves of yellow soybeans contain many

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isoflavonol, isoflavone, pterocarpan, and pheophorbide compounds,5-8 and their extracts can

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exert anti-obesity,9-10 antidiabetic,8,11-12 anti-atherogenic,13 and anti-inflammatory14 effects.

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Extracts of the yellow soybean leaf primarily contain kaempferol glycosides, pterocarpans, and 3


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pheophorbides, which were shown to suppress hyperglycemia through enhancing pancreatic β-

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cell functionality in db/db mice and high-fat diet (HFD)-induced obese diabetic mice.8,15 In

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contrast, the ethanol extract of black soybean leaves (EBL) primarily contains quercetin

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glycosides (QGs), isorhamnetin glycosides (IRGs), and two kaempferol glycosides (KGs), of

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which the latter have also been found in extracts of the yellow soybean leaf.16 Anthocyanins

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isolated from seed coat of black soybeans reduce body weight, serum TG, and cholesterol in

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mice or rats;17-18 however, the biological effects of EBL have not been reported. In this study,

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we investigated the glucose-lowering effect of EBL and studied their molecular mechanisms

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in HFD-fed obese diabetic mice and HepG2 cells. We hypothesized that EBL would promote

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glucose metabolism and diminish hepatic steatosis via effects on adiponectin and its receptor

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signaling in liver.

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MATERIALS AND METHODS Preparation of EBL and Components Analysis. Black soybeans, Glycine max (L.) Merr.

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Seoritae, were collected from JeungPyeong County (Chungcheongbuk-do, Korea) for 107 days.

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The leaves (1.6 kg) after dried were grinded and extracted twice with 70% ethanol (16 L) for

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2 days at room temperature. The extract was filtered and exhaustively concentrated under

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reduced pressure at a temperature below 40 °C to obtain EBL (264 g).

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The EBL components were analyzed and confirmed using a high-performance liquid

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chromatography (HPLC, Shimadzu Corp., Tokyo, Japan) system equipped with a binary pump

76

delivery system, a diode array detector (DAD), an autosampler (Shimadzu Corp.), and a

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Brownlee SPP C18 column (4.6 × 50 mm, 2.7 μm; PerkinElmer Inc., Waltham, MA, USA).

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The samples were injected as 5 μL, and the 0.1% acetic acid (solvent A) and acetonitrile

79

(solvent B) were used as mobile phase. The elution program of linear gradient was as follows: 4



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5–30% B for 0–15 min, 30–80% B for 15–20 min, 80–100% B for 20–23 min, 100% B for 23–

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27.5 min, and 5% B for 27.5–30 min. The flow rate was 1.8 mL/min, and the absorbance

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wavelength was 254 nm. QGs 1, 2, 3, 4, and 5, IRGs 6 and 7, and KGs 8 and 9, which were

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isolated from EBL, were used as external standards for the HPLC analysis (Figure 1A).

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Isoflavones 6''-O-malonyldaidzin (6MD) and 6''-O-malonylgenistin (6MG) were also detected

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from EBL using HPLC–DAD monitoring (Figure 1A).

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Isolation and Identification of Flavonol Glycosides. The Diaion HP-20, Sephadex LH-

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20, and Sep-Pak C18 were used to compound isolation of EBL. Dissolved EBL was separated

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using Diaion HP-20 absorption column chromatography (CC; 9.5 × 20 cm). The 30%, 50%,

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70%, and 100% methanol (MeOH, v/v) orderly eluted column to obtain four fractions (A–D,

90

respectively). Fractions A and B were rich in flavonol glycosides (FGs) as confirmed using

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HPLC–DAD monitoring. Fractions A and B were combined (55.5 g) and separated using ODS

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flash CC (Biotage SNAP Cartridge KP-C18-HS, 9.5 × 15 cm). Elution with aqueous MeOH

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(10%, 20%, 30%, 40%, 50%, and 70% v/v) produced 6 fractions (named as AB-1 to AB-6,

94

respectively). FG-rich fractions (AB-1 and AB-2) were combined (21.0 g) and subsequently

95

purified using Sephadex LH-20 CC (5.0 × 90 cm); elution with 50% MeOH (v/v) yielded 5

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subfractions (named as S-1 to S-5). Fraction S-2 was separated by reversed-phase (RP)

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preparative HPLC (Cosmosil 5C18-MS-II column, 10 × 150 mm, 5 μm; 254 nm; 9% aqueous

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acetonitrile; 4 mL/min) to obtain compounds 1 (6.8 mg) and 2 (5.7 mg). Compound 8 (15.6

99

mg) was separated from fraction S-3 using RP preparative HPLC and eluted with 12%

100

acetonitrile. Fraction S-4 was separated using RP preparative HPLC and eluted with 12%

101

acetonitrile to abtain compound 6 (16.3 mg). Fraction S-5 was subjected to CC on Sep-Pak

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C18 and eluted with 20–30% aqueous MeOH to yield three subfractions (S-5-1 to S-5-3).

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Compounds 3 (40.3 mg) and 4 (34.1 mg) were separated from fraction S-5-1 by RP preparative 5


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HPLC and eluted with 9% acetonitrile. Compounds 5 (11.9 mg) and 6 (22.0 mg) were isolated

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from fraction S-5-2 using RP preparative HPLC and eluted with 11% acetonitrile. Finally,

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compounds 9 (12.9 mg) and 7 (20.2 mg) were separated from fraction S-5-3 by RP preparative

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HPLC and eluted with 11% acetonitrile. Isolated FGs 1–9 (QGs 1‒5, IRGs 6 and 7, and KGs

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8 and 9; Figure 1B) were identified using the basis of spectroscopic data (Supplementary data,

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Figure S1 and Tables S1–S5), and the results showed consistency with previously reported data

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(Supplementary data, References S1–S12). The structures of isoflavones 6MD and 6MG were

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identified based on ESI-MS analysis (Figure S1) and HPLC–DAD monitoring with authentic

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standard compounds of 6MD and 6MG and comparison with previously published data

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(Supplementary data, References S13–S16).

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Animals Experiments. All experiments were approved by the Animal Care and Use

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Committee of Korea Research Institute of Bioscience and Biotechnology (KRIBB, KRIBB-

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AEC-14051). Male 4-week-old C57BL/6J mice were obtained from the Laboratory Animal

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Resource Center of the KRIBB and under controlled temperature (22 ± 2 °C), humidity (50 ±

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5%), and lighting (12 h light/dark cycle) at KRIBB (Daejeon, Korea). The animals were fed a

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10 kcal% normal rodent diet (3.85 kcal/g, 10% fat, 20% protein, 70% carbohydrate; D12450J,

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Supplementary data, Table S6; Research Diets, Inc., New Brunswick, USA) during an

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acclimation period of 3 weeks. Then, the mice were randomly assigned to three groups (n = 10

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per group): a normal diet (ND) group (supplemented with 10 kcal% normal diet); an HFD

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group, which was fed a 60 kcal% diet (5.24 kcal/g, 60% fat, 20% protein, 20% carbohydrate,

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D12492, Supplementary data, Table S6; Research Diets, Inc.) with no supplement; and an

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HFD+EBL group, which was fed HFD with 1% (wt/wt diet) EBL, for 12 weeks.

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Measurement of Biochemical Parameters. The changes of body weight and food intake

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were weekly monitored. The fasting glucose levels were monitored every other week after 12 6



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h of fasting. After 12 weeks, the mice were sacrificed after 12 h of fasting and blood was

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collected into heparin-coated tubes. The plasma was obtained after centrifuged collected blood

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at 800 × g for 15 min at 4 °C and then stored at -70 °C until analysis. The total cholesterol (TC),

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TG, glucose, alanine transaminase (ALT), and aspartate transaminase (AST) were assayed by

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individual available kits (Asan Pharm Co., Seoul, Korea). Glycated hemoglobin (HbA1c)

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levels were measured using an Easy A1c cartridge system (Infopia, Anyang, Korea). Insulin

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and adiponectin levels were measured using an Mouse Insulin ELISA kit (Alpco Diagnostics,

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Salem, NH, USA) and an Mouse Adiponectin/Acrp30 Immunoassay kit (R&D Systems, Inc.,

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Minneapolis, MN, USA), respectively. Non-esterified fatty acids (NEFAs) were measured by

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an automated blood chemistry analyzer (Hitachi-7150, Hitachi Medical, Tokyo, Japan).

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Histopathological Analysis of the Liver and Adipose Tissue. Liver or epididymal

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adipose tissue samples were fixed in a buffer solution containing 10% formalin and processed

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for paraffin embedding. Then, samples were cut into 4-µm sections and stained with

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hematoxylin-eosin (H&E). Images of stained liver or adipose tissue were obtained using an

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Olympus BX61 microscope system equipped with Olympus DP71 digital camera (Tokyo,

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Japan). The dimension of adipocytes were detected by the MetaMorph® Image Analysis

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Software (Molecular Devices, Sunnyvale, CA, USA).

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Measurement of Liver Lipid Contents and AMPK Activity. Hepatic TG and TC

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contents were measured using the Folch et al. reported methods.19 The activity of hepatic

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AMPK was measured using an AMPK assay kit (CycLex Co., Ltd., Ina, Japan) in accordance

148

with the manufacturer’s recommendations.

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Real-Time Quantitative RT-PCR (qRT-PCR). The removed liver and fat tissue were

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socked in RNAlater solution (Qiagen, Valencia, CA, USA) for RNA extraction. Afterward,

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TRI reagent (Ambion, CA, USA) was used to obtain the RNA layer from liver or adipose tissue. 7


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Then an RNeasy mini kit (Qiagen, Valencia, CA, USA) was used to isolation of total RNA.

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The total RNA was used to synthesis of cDNA by High-Capacity cDNA Reverse Transcription

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kit (Thermo Fisher Scientific, Inc., MA, USA). A 7500 real-time PCR system (Applied

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Biosystems) was used to conducting Real-time quantitative polymerase chain reaction (qRT-

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PCR) for quantification of RNA expression with the FastStart Universal SYBR Green Master

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(Roche, Mannheim, Germany). The used primer sequences are listed in supplementary Table

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S7.

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Immunoprecipitation and Immunoblotting. Animal tissue proteins and cell proteins

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were subjected to immunoprecipitation (IP) and immunoblotting (IB). For IP of IRS and AKT,

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1 mg total protein was incubated with specific antibodies against IRS and AKT, respectively,

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using a magnetic beads system (Bio-Red Laboratories, Inc., Japan). Phosphorylated or total

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protein expression was analyzed by IB with antibodies against pAMPK, AMPK, pIRS, IRS,

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pAKT, AKT, and GAPDH. Immunoreactive protein expression was measured using an

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enhanced chemiluminescence western blot kit (Thermo Fisher Scientific, Inc.) and an LAS-

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4000 luminescent image analyzer (Fuji Photo Film, Tokyo, Japan). Intensities of bands were

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quantified by the Fujifilm Image MultiGauge software (Fuji Photo Film). GAPDH was used

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as a loading control.

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Oral Glucose Tolerance Test. Male C57BL/6J (7-week-old) mice were fed ND (10 kcal%;

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Research Diets, Inc.) or HFD (60 kcal%; Research Diets, Inc.) for 8 weeks, and then the HFD-

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fed mice were randomly assigned to two groups (n = 5 per group): an HFD group, with daily

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gavage of the vehicle as a control, and an EBL-fed group (500 mg·kg−1·day−1, orally). EBL

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was suspended in sterile purified water containing 10% polyethylene glycol and 0.5% Tween

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80. Oral glucose tolerance test (OGTT) was conducted after 17 days of oral gavage and fasted

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for 16 h. Afterward mice were orally administered glucose (2 g/kg body weight) and blood was 8



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collected and measured glucose levels from the tail vein blood at 0, 15, 30, 60, 90, and 120 min

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after glucose administration. The glucose levels were measured using an Accu-Chek Active

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glucometer (Roche, Mannheim, Germany).

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HepG2 Cell Culture and Measurement of TG Contents. The HepG2 human

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hepatocellular carcinoma cell line was purchased from the American Type Culture Collection

181

(Manassas, VA, USA). HepG2 cells were cultured in low-glucose Dulbecco's modified Eagle's

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medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100

183

μg/mL streptomycin and incubated in 5% CO2 at 37 °C.

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The palmitic acid (PA) induction medium was prepared using the Mayer et al. reported

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methods.20 Lipid accumulation was induced with 0.2 mM PA, with or without sample, for 24

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h. First, a 100 mM PA solution was prepared in sterile purified water by heating to 70 °C with

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vortexing until PA was dissolved. Immediately afterward, the 100 mM PA solution was diluted

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to 0.2 mM PA inducing serum-free low-glucose DMEM medium containing 3% fatty acid-free

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bovine serum albumin (BSA). The 0.2 mM PA solution was shaken at 37 °C for 2 h and then

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immediately used to cell experiments. Serum-free low-glucose DMEM containing 3% fatty

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acid-free BSA was used as a vehicle control. Following treatment, total cell protein extract was

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collected using a cell lysis reagent (Sigma) and 1% Triton X-100 (Sigma). The TG content was

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assayed by a TG kit (Asan Pharm Co.).

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Statistical Analyses. Results from the animal experiments are presented as the mean ±

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standard error (SE). Statistical differences were determined using one-way analysis of variance

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using the JMP® statistical software (SAS Institute, Cary, NC, USA). Results from the HepG2

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cell experiments were calculated as the mean ± standard deviation (SD) and analyzed using the

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Student’s t-test to identify significant differences between the test groups and the control group.

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P < 0.05 was considered significant. 9


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RESULTS

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HPLC Analysis of EBL. EBL was analyzed by HPLC–DAD at 254 nm (Figure 1A). It

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primarily included QGs, IRGs, and KGs. The five QGs were quercetin 3-O-β-D-

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glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-galactopyranoside (1), quercetin

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3-O-β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside

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quercetin 3-O-(2-β-D-glucopyranosyl)-β-D-galactopyranoside (3), quercetin 3-O-(2-β-D-

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glucopyranosyl)-β-D-glucopyranoside (4), and quercetin 3-O-(2-α-D-rhamnopyranosyl-

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(1→2)-β-D-galactopyranoside (5). The two

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glucopyranosyl)-β-D-galactopyranoside (6) and isorhamnetin 3-O-α-L-rhamnopyranosyl-

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(1→2)]-β-D-galactopyranoside (7), and the two KGs were kaempferol 3-O-(2,6-di-O-α-L-

211

rhamnopyranosyl]-β-D-galactopyranoside (8) and kaempferol 3-O-α-L-rhamnopyranosyl-

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(1→2)-β-D-galactopyronoside (9) (Figure 1B). Compounds 8 and 9 have also been found in

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the extracts of the yellow soybean leaf.8 Compounds 3 and 4 were the most abundant QGs in

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EBL, based on comparisons of the peak areas with those of standard compounds (Figure 1A).

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QG3 and QG4 were determined and quantified by HPLC–DAD, by comparison to their

216

standard curves. QG3 and QG4 were quantified as 24.56 ± 0.38 μg/mg EBL and 15.23 ± 0.11

217

μg/mg EBL, respectively.

IRGs were

isorhamnetin

(2),

3-O-(2-β-D-

218

EBL Reduced Body Weight and Glucose Levels. The body weights did not different

219

among the all groups at initial of this study. During the 12-week feeding period, the mice in the

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three groups continued to increase in body weight, although to different extents (Figure 2A).

221

After 2 weeks, the mean body weight of the HFD group (31.2 g) was significantly increased

222

compare to that of the ND group (27.2 g). Supplementation of EBL significantly reduced body

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weight (31.4 g) compared to that of the HFD group (35.0 g), starting from 4 weeks. After 12 10



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weeks, the body weight gain of the HFD group was 1.9-fold of that in the ND group.

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Supplementation of the HFD with EBL significantly lowered the body weight gain, by 17.8%

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(Table 1). The food and energy intake were similar in the two groups receiving HFD. At the

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end of the experiment, the organ weight including liver, pancreas, muscle, and epididymal fat

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tissue were measured. EBL significantly lowered the HFD-induced increase in liver weight

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(Table 1).

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The HFD-fed mice developed hyperglycemia and hyperlipidemia, as confirmed based on

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their plasma profiles at 12 weeks (Table 2). The plasma levels of fasting glucose, HbA1c, and

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insulin were significantly higher in the HFD group than in the ND group. However, the plasma

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glucose and HbA1c levels were significantly lower in the HFD+EBL group, by 27.3% and

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8.7%, respectively, than in the HFD group. From 2 weeks onwards, the glucose levels of the

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HFD+EBL group were markedly lower than those of the HFD group (Figure 2B). The mean

236

plasma insulin concentration of the HFD+EBL group was significantly lower, by 73.5%, than

237

that of the HFD group. The homeostatic model assessment of insulin resistance (HOMA-IR)

238

index was dramatically increased in the HFD group than in the ND group. Administration of

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EBL significantly decreased the HOMA-IR index, by 76.2%, compared with that of the HFD

240

group. The plasma TG, TC, and NEFA levels were markedly lower, by 45.9%, 12.9%, and

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15.8%, respectively, in the EBL group than in the HFD group. The plasma AST and ALT levels,

242

parameters of liver function, were lowered by 9.9% and 29.1%, respectively, in the HFD+EBL

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group compared to those of HFD group, with ALT showing a significant difference between

244

the groups. Overall, these data suggest that EBL protected HFD-fed mice against

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hyperglycemia and hyperlipidemia.

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EBL Suppressed Hepatic Steatosis and Improved AMPK Activity. Next, histology

247

was performed and the lipid contents of the liver were determined. The number and volume of 11


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hepatic lipid droplets in the HFD group markedly increased compared to that in the ND group,

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indicating hepatic steatosis (Figure 2C). However, the number and volume of lipid droplets

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markedly decreased in the EBL group. EBL supplementation significantly lowered hepatic TG

251

and TC contents compare to those in the HFD group (Figure 2D and E).

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To elucidate the mechanism responsible for controlling hepatic steatosis, the activity of

253

hepatic AMPK, an important regulator of energy metabolism, was measured, and it was found

254

that AMPK activity significantly increased in the EBL group compared to that in the HFD

255

group (Figure 2F).

256

The expression of adiponectin decreased in obesity induced by HFD. In the present study,

257

the plasma adiponectin levels reverted to normal by supplementing the HFD with EBL (Figure

258

2G). These results indicated that EBL treatment suppressed hepatic steatosis by AMPK

259

activation, and this effect might be related to adiponectin expression.

260

EBL Restored the Expression of Adiponectin and Its Downstream Genes/Proteins.

261

For understanding the molecular mechanisms responsible for the action of EBL, a number of

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metabolism-related protein and mRNA expression were measured. The mRNA expression

263

patterns of adiponectin in adipose tissue corresponded to those of its plasma levels (Figure 3A).

264

Expression of the adiponectin receptors decreased in the liver of the HFD-fed mice. However,

265

the mRNA expression of AdipoR1 and AdipoR2 were significantly increased in the EBL

266

treatment group (Figure 3B). AdipoR2 is a highly liver-specific isoform. Adiponectin

267

activation of receptor signaling can reduce hepatic steatosis through activation of AMPK or

268

fatty acid oxidation. The expression levels of transcription factors from the PPAR family

269

related to lipid metabolism and their coactivator, PPARγ coactivator-1 (PGC-1), were

270

modulated by EBL treatment. EBL administration reversed the HFD-induced decrease in

271

PPARα, PPARδ, and PGC-1, and the HFD-induced increase in the PPARγ expression levels 12



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(Figure 3C). The mRNA expression of acetyl-coenzyme A carboxylase 1 (ACC1), ACC2, fatty

273

acid synthase (FAS), and carnitine palmitoyltransferase (CPT)-1α, downstream proteins of the

274

PPAR family, was also restored in the HFD+EBL group (Figure 3D). Furthermore, the mRNA

275

expression of insulin sensitivity-related proteins, including glucose transporter (GLUT)-2 and

276

IRS-2, increased in the HFD+EBL group (Figure 3E). Additionally, expression levels of

277

gluconeogenesis-related transcription factors, forkhead box protein O1 (FOXO1) and FOXA2,

278

decreased in the HFD+EBL group. The protein expression of phosphorylated AMPK, IRS, and

279

AKT also increased in the EBL group compared to the levels in the HFD group (Figure 3F).

280

These results indicated that EBL inhibited HFD-induced hepatic steatosis through adiponectin

281

receptor signaling, which, in turn, suppressed lipogenesis, fat oxidation, and insulin sensitivity.

282

EBL Increased Insulin Sensitivity-Related Gene Expression in Adipose Tissue. In

283

the histological analyses, feeding of HFD increased the size of adipocytes in epididymal

284

adipose tissue (Figure 4A and B). EBL supplementation significantly suppressed the HFD-

285

induced size of adipocytes. Next, the mRNA expression of insulin sensitivity-related genes was

286

determined. The mRNA expression levels of insulin receptor (IR), IRS-1, and GLUT-4 were

287

increased in the HFD+EBL group as compared with those in the HFD group (Figure 4C). These

288

results suggested that EBL may enhance insulin sensitivity in insulin target tissues like the

289

epididymal adipose tissue.

290

Therapeutic Effect of EBL in OGTT. To determine the therapeutic effect of EBL on

291

glucose tolerance using OGTT, the HFD was fed for 8 weeks to induce impaired glucose

292

tolerance. Then, randomly assigned mice were orally administered EBL or vehicle for 17 days.

293

In ND-fed mice, the blood glucose level peaked at 15 min after glucose administration, whereas

294

in both the HFD and HFD+EBL groups, the glucose level peaks shifted to 30 min and longer;

295

thereafter, glucose was gradually eliminated until 120 min (Figure 5A). However, glucose 13


Page 15 of 45


296

elimination was greater in the HFD+EBL group at 60 and 90 min. The calculated area under

297

the glucose concentration–time curve values showed that the elimination of glucose was

298

dramatically accelerated in the HFD+EBL group compared to the HFD group (Figure 4B).

299

These results indicate that EBL can therapeutically diminish insulin resistance in HFD-fed mice.

300

QGs and IRGs Reduced TG Accumulation in HepG2 Cells. To determine whether the

301

purified constituents of EBL could directly inhibit excessive lipid accumulation through

302

activation of AMPK in hepatocytes, effects of QGs, IRGs, and KGs on intracellular TG

303

accumulation and AMPK activation were examined in HepG2 cells. Treatment with 100 μM

304

QG3, QG5, or quercetin significantly reduced the intracellular TG accumulation and increased

305

phosphorylation of AMPK in PA-induced HepG2 cells (Figure 6A and B). Treatment with 50

306

μM IRG6 or IRG7 also significantly decreased the PA-induced TG accumulation (by 13.9%

307

and 21.6%, respectively) in HepG2 cells (Figure 6A). Treatment with 100 μM KG8 or KG9

308

did not change PA-induced TG accumulation (data not shown). The phosphorylation form of

309

AMPK was markedly increased by treatment with IRG7 or isorhamnetin but not with IRG6

310

(Figure 6C). To confirm the effectiveness of the compounds, various concentrations of QG3,

311

QG5, and IRG7 were used to treat the cells, and the data showed that the compounds

312

significantly increased phosphorylation of AMPK and AKT in a dose-dependent manner

313

(Figure 6D and E). These results suggest that the components of EBL can directly reduce

314

hepatic lipid accumulation through enhancing the activities of the AMPK/AKT pathway.

315 316

DISCUSSION

317

Our previous studies have revealed that leaf extracts of yellow soybean, containing

318

kaempferol glycosides, pheophorbides, and pterocarpans, suppress hyperglycemia trough

319

enhancing pancreatic β-cell functionality and insulin sensitivity in adipose tissue of db/db mice 14



Page 16 of 45

320

and HFD-induced diabetic mice.8,15 In this study, we found that EBL, unlike yellow soybean

321

leaf extracts, mainly contains QGs and IRGs (Figure 1A and B). Quercetin is the most common

322

natural flavonoid; it primarily exists in its glycoside form. Quercetin exhibits antidiabetic

323

effects by enhancing insulin secretion of β-cell function21 and restoring insulin signaling.22 It

324

has also shown anti-inflammatory effects.23 Quercitrin, isoquercitrin, and quercetin-3-

325

glucuronide, are natural quercetin glycosides and have also been reported to show biological

326

activities against hyperglycemia and inflammation.23 Compared to quercetin, quercetin-3-

327

glucuronide has been shown to be less effective at inhibiting inflammatory gene expression in

328

lipopolysaccharide-induced RAW264.7 macrophages. Alternatively, treatment with rutin, a

329

quercetin glycoside, lowered serum TG and TC levels in streptozotocin-induced diabetic rats

330

more effectively than quercetin.24 Additionally, rutin was investigated for its antioxidant and

331

hepatoprotective activities.25 Isorhamnetin, a 3'-methoxylated derivative of quercetin, showed

332

hepatoprotective, anti-adipogenic, and anti-inflammatory effects.26-27 Opuntia ficus-indica

333

(OFI) extracts, which are rich in IRGs, reduced proinflammatory mediators including nitric

334

oxide, tumor necrosis factor α, cyclooxygenase-2 (COX-2), and interleukin-6, in vitro and in

335

vivo.28 IRGs-rich OFI, but the major IRG constituents were different from EBL, also reversed

336

HFD-induced body weight gain, and the associated increased glucose and insulin levels, by

337

improving hepatic insulin sensitivity and reducing hepatic lipid contents through ameliorating

338

endoplasmic reticulum stress and fatty acid oxidation.29 However, biological activities against

339

obesity or hyperglycemia have not been previously reported for the EBL and the QGs and IRGs

340

identified in this study.

341

EBL, containing QGs and IRGs, reduced the HFD-induced obesity and hyperglycemia,

342

and improved plasma glucose homeostasis and lipid metabolism. These effects of EBL may be

343

due to its ability to reverse hepatic steatosis through AMPK activation by adiponectin. 15


Page 17 of 45


344

Therefore, our subsequent studies focused on elucidating the adiponectin-related molecular

345

mechanism of EBL action. A 30-kDa adiponectin is released by adipocytes and circulates as 3

346

molecular forms: full-length trimer adiponectin (90 kDa); 6 molecules comprised low-

347

molecular-weight (LMW) adiponectin (180 kDa); 12 or 18 molecules comprised high-

348

molecular-weight (HMW) adiponectin (360–540 kDa).30 Full-length trimer adiponectin, HMW

349

and LMW adiponectin are stable under basic conditions in vitro and in vivo.31 However, these

350

complexes can be cleaved to full-length trimer adiponectin or globular-domain trimers under

351

acidic conditions (below pH 7).32 Full-length trimer adiponectin bind to AdipoR1 and AdipoR2,

352

whereas HMW and LMW adiponectin bind to T-cadherin.33-34 Full-length trimer adiponectin

353

showed a greater glucose-lowering effect than HMW and LMW adiponectin in mice.31

354

Adiponectin exerts an antisteatotic activity by binding to AdipoR1 and AdipoR2 to initiate

355

their signaling. The intracellular domains of AdipoR1 (amino acids 4–142) and AdipoR2

356

(amino acids 4-136) bind to adaptor protein, phosphotyrosine interacting with PH domain and

357

leucine zipper 1 (APPL1)35 to facilitate liver kinase B1 (LKB1)-induced activation of AMPK.36

358

Alternatively, adiponectin enhances the activity of AMPK by a Ca2+/calmodulin-dependent

359

protein kinase kinase, independent of the APPL1/LKB1 pathway.36 Adiponectin-activated

360

AMPK stimulates glucose uptake and fat ultilization in the muscle while suppressing

361

gluconeogenesis in the liver.37-38 Supplementation with EBL significantly increased the plasma

362

adiponectin level and adiponectin mRNA expression in the adipose tissue of HFD-fed mice.

363

The mRNA levels of AdipoR1 and AdipoR2 increased, accompanied by AMPK activation, in

364

the HFD+EBL group.

365

Activated AdipoR2, but not AdipoR1, increases the expression of PPARα-mediated β-

366

oxidation-related genes in the liver.39 Other than PPARα, other members of the PPAR family

367

also play pivotal roles in hepatic lipid metabolism. PPARδ ameliorates hepatic insulin 16



Page 18 of 45

368

resistance by decreasing gluconeogenesis through downregulation of FOXO1, whereas PPARγ

369

activation increases hepatic lipid storage through regulation of its target genes. EBL

370

upregulated PPARα, PPARδ, and their coactivator PGC-1. Subsequently, treatment with EBL

371

increased β-oxidation-related CPT-1α and decreased gluconeogenesis-related FOXO1 and

372

FOXA2. The HFD+EBL group showed improvement in insulin sensitivity through an increase

373

in GLUT-2 and IRS-2 gene expression and activation of AKT. Furthermore, treatment with EBL

374

decreased the lipogenesis-related PPARγ, ACC1, ACC2, and FAS expression. These results

375

indicated that the intake of EBL ameliorates glucose homeostasis and hepatic steatosis through

376

activation of adiponectin receptor signaling in HFD-fed mice. Additionally, the QG3, QG5,

377

and IRG7 constituents of EBL directly reversed the PA-induced hepatic steatosis through

378

enhancing the activity of AMPK in HepG2 cells. Our results revealed that EBL and its active

379

constituents may have an enhanced effect in vivo and in vitro by affecting adiponectin-mediated

380

receptor signaling and directly activating AMPK. Further studies are needed to clarify that EBL

381

enhanced adiponectin receptor signaling and the crosstalk with direct activation of AMPK in

382

liver. However, data from this study has investigated EBL as a valid material in HFD-induced

383

hyperglycemia.

384

In summary, present study revealed, for the first time, the antidiabetic and anti-obesity

385

effects of EBL and identified its active constituents. EBL reduced body weight gain and

386

improved glucose homeostasis; it lowered glucose, insulin, HbA1c, and HOMA-IR index

387

levels in HFD-fed mice. These effects may be due to improved hepatic function associated with

388

enhanced adiponectin receptor signaling. EBL increased plasma adiponectin levels, which led

389

to enhancing the activities of AdipoR1 and AdipoR2 and subsequently activation of AMPK

390

and PPARs (Figure 7). This in turn led to the upregulation of β-oxidation- and insulin

391

sensitivity-related genes and proteins, and the downregulation of hepatic lipogenesis- and 17


Page 19 of 45


392

gluconeogenesis-related genes. Overall, EBL suppressed hyperglycemia and hepatic steatosis

393

via adiponectin and AMPK signaling. Importantly, QGs and IRGs were identified as active

394

components of EBL. QG3, QG5, and IRG7 directly reduced lipid accumulation in HepG2 cells

395

through enhancing the activity of AMPK. This direct suppression of lipogenesis in hepatocytes

396

may contribute to the action of EBL in vivo. Taken together, leaves of black soybean can be a

397

good basis for the progression of health food targeting for antidiabetic and anti-obesity.

398 399

Supporting Information

400

Chemical structure elucidation of flavonol glycosides 1‒9 (Tables S1‒S5 and Figure

401

S1); the composition of experimental diets (Table S6); and primer sequences used for real-time

402

qRT-PCR (Table S7).

403 404

Funding

405

This work was supported by the High Value-added Food Technology Development Program

406

(No. 313044-3) of the Ministry of Agriculture, Food and Rural Affairs and the KRIBB

407

Research Initiative Program (KGM2131824 and KGS1001815), Republic of Korea.

408 409

Notes

410

The authors declare no conflicts of interest.

411 412

18



413

Page 20 of 45

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24



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552

Figure legends

553

Figure 1. HPLC profile of EBL and the chemical structures of the main components. (A) HPLC

554

chromatograms of EBL were detected at 254 nm. (B) Chemical structures of QGs (1-5), IRGs

555

(6 and 7), and KGs (8 and 9) isolated from EBL. 6MD: 6''-O-malonyldaidzin; 6MG: 6''-O-

556

malonylgenistin.

557 558

Figure 2. Effects of EBL supplementation on body weight gain (A) and fasting glucose levels

559

(after 12 h fasting; B) in HFD-fed mice. (C) Histology of the livers stained with H&E. (D)

560

Hepatic TG and (E) TC contents. (F) Relative AMPK activity in the liver. (G) Plasma

561

adiponectin levels. Values are presented as mean ± SE.

562

are significantly different at P < 0.05.

a,b,c

Different letters within a variable

563 564

Figure 3. Effects of EBL supplementation on hepatic mRNA and protein expression in HFD-

565

fed mice. (A) Relative mRNA expression levels in the adipose tissue. (B-E) Relative mRNA

566

expression levels in the liver. The mRNA levels were measured by real-time qRT-PCR and

567

normalized using GAPDH as a reference gene. (F) Western blot of hepatic AMPK, IRS, and

568

AKT detected with specific antibodies. Values are presented as mean ± SE. a,b,c Different letters

569

within a variable are significantly different at P < 0.05.

570 571

Figure 4. Effects of EBL supplementation on insulin sensitivity of epididymal adipose tissue.

572

(A) Histology of the epididymal adipose tissue stained with H&E. (B) Quantitative

573

measurement of adipocyte size. (C) Relative mRNA expression levels in the adipose tissue.

574

The mRNA levels were measured by real-time qRT-PCR and normalized using GAPDH as a

575

reference gene. Values are presented as mean ± SE.

a,b,c

Different letters within a variable are 25


Page 27 of 45

576


significantly different at P < 0.05.

577 578

Figure 5. Effects of EBL supplementation on OGTT in HFD-fed mice. The OGTT was

579

performed on day 17 after the start of the oral gavage of 500 mg·kg−1·day−1 EBL in HFD-

580

induced mice. (A) Changes in glucose levels after glucose (2 g/kg body weight) administration.

581

Blood glucose level was measured in tail vein blood at 0, 15, 30, 60, 90, and 120 min. (B) Area

582

under the curve (AUC) of plasma glucose during OGTT. Values are presented as mean ± SE.

583

a,b,c

Different letters within a variable are significantly different at P < 0.05.

584 585

Figure 6. Effects of EBL components on lipid accumulation in PA-induced HepG2 cells. (A)

586

Effects of QGs and IRGs on TG accumulation and (B, C) on AMPK activation in PA-induced

587

HepG2 cells. (D, E) Effects of QG3, QG5, and IRG7 on AMPK and AKT activation in PA-

588

induced HepG2 cells. Western blot of AMPK and AKT detected with specific antibodies.

589

Values are presented as mean ± SD. #P < 0.01 versus cells treated with media only; *P < 0.05,

590

** P < 0.01 versus cells treated with PA only.

591 592

Figure 7. The possible molecular mechanisms of anti-diabetic effect by EBL, QGs, and IRGs.

593

EBL suppressed hyperglycemia and hepatic lipid accumulation in HFD-fed mice by enhancing

594

adiponectin receptor signaling and accompanied with activation of AMPK.

26



Page 28 of 45

Table 1. Effects of EBL supplementation on body and organ weights in HFD-fed mice ND

HFD

HFD+EBL

12.5 ± 1.1c

23.6 ± 0.5a

19.4 ± 1.3b

Food intake (g/day)

2.4 ± 0.1a

2.1 ± 0.1b

1.9 ± 0.1b

Energy intake (kcal/day)

9.1 ± 0.2b

11.0 ± 0.4a

10.3 ± 0.2a

Liver (g)

1.29 ± 0.05b

1.56 ± 0.10a

1.12 ± 0.04b

Pancreas (g)

0.19 ± 0.01a

0.23 ± 0.03a

0.21 ± 0.01a

Muscle (g)

0.29 ± 0.01b

0.32 ± 0.01a

0.34 ± 0.00a

Fat tissues (g)

0.60 ± 0.06b

1.06 ± 0.07a

0.98 ± 0.10a

Weight gain (g)

Values are presented as mean ± SE, n = 10. a,b Means not sharing a common letter are significantly different between groups (P < 0.05).

27


Page 29 of 45


Table 2. Effects of EBL supplementation on plasma profiles in HFD-fed mice ND

HFD b

HbA1c (%)

4.4 ± 0.0

Insulin (ng/mL)

0.3 ± 0.0

HOMA-IR

1.8 ± 0.4

TG (mg/dL)

90.7 ± 11.9 111.3 ± 2.6

HDL/TC (%)

52.3 ± 5.4

NEFA (mEq/L)

1.7 ± 0.2

AST (IU/L)

56.2 ± 1.3

ALT (IU/L)

33.5 ± 1.3

4.6 ± 0.1

c

3.4 ± 0.3

c

b

TC (mg/dL)

a

b b b

HFD+EBL

28.6 ± 3.4 b

a a

a

42.6 ± 3.8

b

1.9 ± 0.1 69.8 ± 2.7 40.2 ± 1.9

0.9 ± 0.2

a

117.1 ± 17.5 145.5 ± 3.9

4.2 ± 0.0

a a a

6.8 ± 1.6 a

b b b

63.3 ± 6.6

c

126.7 ± 9.3 55.6 ± 1.0 1.6 ± 0.1

ab

a

b

62.9 ± 2.3 28.5 ± 1.1

ab

c

Values are presented as mean ± SE, n = 10. a,b,c Means not sharing a common letter are significantly different between groups (P < 0.05).

28


Fig. 1


Page 30 of 45

Absorbance (mAU at 254 nm)

A 3 750

4 500

8 250

5 1

2

69 7

6MG

6MD

0

5.0

7.5

10.0

12.5

Retention time (min)


15.0

17.5

20.0

Page 311of (continued) 45 Fig.


B

2

1

6

4

3

7


8

5

9

Fig. 2


A

Page 32 of 45

55 ND

HFD

50

a

HFD+EBL

a

Body weight (g)

45

a

a

a

a

b

a

40 a 35

a b

a b b

ab

ab b

25

b

b

11

b

b

a 30

b

b

b

b

4

5

b

b

b

b

8

9

10

c

b

b

20 0

1

2

3

6

7

Feeding duration (weeks)


12


B


250 ND a

Fasting glucose (mg/dL)

HFD 200

a

HFD+EBL a

a

a

b

b

150 b b

b c

100

c

b c

c

50

0 0

2

4

8

Feeding duration (weeks)


10

12

Fig. 2 (continued)


C

E

60 a

50 40

HFD+EBL

HFD

b b

30 20 10 0

Hepatic TC (mg/g liver)

Hepatic TG (mg/g liver)

D

ND

Page 34 of 45

1.6

a ab b

1.2

0.8

0.4

0.0 ND

HFD

HFD+EBL ACS Paragon Plus Environment

ND

HFD

HFD+EBL


G 14

3.0 a 2.5 2.0 ab 1.5

b 1.0 0.5 0.0

plasma adiponectin (μg/mL)

AMPK activity (relative fold)

F


a

a

12 b 10 8 6 4 2 0

ND

HFD

HFD+EBL


ND

HFD

HFD+EBL

A


B

2.5

Page 36 of 45

2.0

2.0

a

a

mRNA expression (fold)


ND

1.5 b

1.0

0.5

HFD

EBL HFD+EBL

a

1.5

a

a ab b

b

AdipoR1

AdipoR2

1.0

0.5

0.0

0.0 Adiponectin ND HFDEBL

C a

3.0


Fig. 3

ND a

HFD

EBL HFD+EBL

a

2.0

b

ab

a

a

b

a

b

1.0

b

b


0.0 PGC-1

PPARα

PPARγ

PPARδ

D


2.5


ND

HFD+EBL EBL

HFD

a

a

2.0

1.5 b

a

a

a

1.0 b

b

b b

b

0.5

c

0.0 ACC1

E

ACC2

FAS

CPT-1α

4.0 ND



HFD

EBL HFD+EBL

a

3.0 ab

a 2.0 b a

b b

1.0

b

ab

a

b b


0.0 GLUT-2

FOXO1

FOXA2

IRS-2

Fig. 3 (continued)


F

Page 38 of 45

ND

HFD

HFD+EBL

2.7

1.0

2.6

2.2

1.0

2.8

IB: pAMPK IB: AMPK pAMPK/AMPK IP: IRS IB: pIRS IP: IRS IB: IRS pIRS/IRS IP: AKT IB: pAKT IP: AKT IB: AKT pAKT/AKT

1.4

1.0

IB: GAPDH


1.7

Page Fig.394of 45


A

B

HFD

HFD+EBL

Size of adipocytes (µm2)

ND

14,000 a

12,000

b

10,000 c

8,000 6,000 4,000 2,000 0

ND


HFD

HFD+EBL

Fig. 4 (continued)


C


2.5

ND

HFD

Page 40 of 45

HFD+EBL a

a

2.0

a ab ab

a

1.5 ab

b

b

b

b

IRS-1

IRS-2

b

1.0

0.5

0.0 IR


GLUT-4

Page Fig.415of 45


B

500

AUC of glucose level (mg•min/dL)

A

ND

Blood glucose (mg/dL)

a

HFD

400

HFD+EBL

a

a a

300

a b

b

a

b

200

b c 100

c

25,000

a 20,000

b

15,000

c 10,000

5,000

0

0 0

15

30 60 Time (min)

90

120


ND

HFD

HFD+EBL


A

B

Page 42 of 45

120

Intracellular TG (% of control)

Fig. 6

#

100

*

**

80

**

** **

60

**

40 20

0 PA (0.2 mM)

-

+

+

+

+

+

+

+

+

+

+

Samples

-

-

QG1

QG2

QG3

QG4

QG5

Q

IRG6

IRG7

IR

Conc. (μM)

0

0

100

100

100

100

100

100

50

50

50

PA (0.2 mM)

-

-

+

+

+

+

+

+

+

insulin (0.2 μM)

-

+

+

+

+

+

+

+

+

QGs (100 μM)

-

-

-

QG1

QG2

QG3

QG4

QG5

Q

1.3

1.8

1.9

1.4

1.6

1.1

pAMPK AMPK pAMPK/AMPK


1.0

1.0

1.0



C

PA (0.2 mM)

-

-

+

+

+

+

Insulin (0.2 μM)

-

+

+

+

+

+

IRG (50 μM)

-

-

-

IRG6

IRG7

0.7

1.5

1.0

1.0

1.3

IR

pAMPK

AMPK pAMPK/AMPK


1.2

Fig. 6 (continued)


D

Page 44 of 45

PA (0.2 mM) Insulin (0.2 μM)

-

+

+ +

+ +

+ +

+ +

+ +

QG3 (μM)

-

-

-

50

100

-

-

QG5 (μM)

-

-

-

-

-

50

100

1.6

1.0

1.1

1.4

1.1

1.4

2.0

1.3

1.7


0.8

pAKT AKT pAKT/AKT

E

0.1

2.0

1.0

1.5

PA (0.2 mM)

-

-

+

+

+

+

Insulin (0.2 μM)

-

+

+

+

+

+

IRG7 (μM)

-

-

-

20

50

100

1.1

2.1

1.0

1.0

1.4

2.0


pAKT AKT ACS Paragon Plus Environment

pAKT/AKT

0.2

2.1

1.0

1.3

1.3

1.6

Page 457of 45 Fig.


Blood

Liver

AdipoR1

AdipoR2

Adipose tissue

p AMPK IRS

p

p AKT

Insulin sensitivity

PPARα, PPARδ, PPARγ CPT-1, ACCs, FAS

TG

Adiponectin Insulin sensitivity

Lipid accumulation

Glucose GLUT-2

EBL QGs, IRGs Adiponectin TG Glucose


Black Soybean Leaf Extract Suppresses Hyperglycemia and Hepatic

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