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Pectin lyase-modified red ginseng extract improves glucose homeostasis in high fat diet-fed mice
Journal Pre-proof Pectin lyase-modified red ginseng extract improves glucose homeostasis in high fat diet-fed mice Go Woon Kim, Mi-Kyung Pyo, Sung Hyun Chung PII:
S0378-8741(19)32743-6
DOI:
https://doi.org/10.1016/j.jep.2019.112384
Reference:
JEP 112384
To appear in:
Journal of Ethnopharmacology
Received Date: 9 July 2019 Revised Date:
31 October 2019
Accepted Date: 10 November 2019
Please cite this article as: Kim, G.W., Pyo, M.-K., Chung, S.H., Pectin lyase-modified red ginseng extract improves glucose homeostasis in high fat diet-fed mice, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112384. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
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Pectin lyase-modified red ginseng extract improves glucose homeostasis in
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high fat diet-fed mice
3 Go Woon Kim1, Mi-Kyung Pyo2, Sung Hyun Chung1*
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Department of Pharmacology, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro,
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Seoul 02447, Republic of Korea 2
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International Ginseng and Herb Research Institute, Geumsan, Republic of Korea
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Emails of authors:
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Kim, Go Woon: [email protected];
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Pyo, Mi-Kyung: [email protected];
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Chung, Sung Hyun: [email protected]
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*
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Department of Pharmacology, Kyung Hee University, 26 Kyungheedae-ro, Seoul 02447, Republic of
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Korea
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Tel: +82-2-961-0373
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E-mail: [email protected]
Corresponding author: Sung Hyun Chung, Ph.D.
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Abstract
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Ethnopharmacological relevance: Red ginseng has long been used as a traditional folk medicine for
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various diseases including diabetes. Recently, a preparation of red ginseng extract by pectin lyase
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modification has been developed and named as GS-E3D.
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Aim of the study: The aim of this study is to evaluate the preventive effect of GS-E3D on
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hyperglycemia induced by feeding a high fat diet (HFD) in mice.
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Materials and Methods: GS-E3D was orally administered to C57BL/6J mice at different doses (250,
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500, or 1000 mg/kg/day) for 6 weeks while on a HFD. Body weight and blood glucose were
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monitored weekly, and oral glucose tolerance test (OGTT) was performed at 5th week of the
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experiment. Glycemic indications and metabolic parameters were further measured in serum.
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Results: Six weeks of GS-E3D treatment to mice significantly inhibited HFD-induced body weight
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gain, hyperglycemia, hyperinsulinemia and hypertriglyceridemia. Notably, GS-E3D treated mice at
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doses of 250, 500 and 1000 mg/kg showed 41.8%, 45.0% and 55.1% reduction in insulin resistance
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index, respectively, compared to HFD control mice. OGTT revealed that GS-E3D markedly prevented
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steep rise of blood glucose and insulin levels after glucose challenge and ameliorated HFD-induced
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glucose and insulin intolerance. The histological analysis showed enlarged adipocytes in HFD-fed
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mice whereas the adipocyte hypertrophy was prevented in GS-E3D treated mice in a dose-dependent
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manner. Furthermore, when peripheral glucose uptake level was assessed by total and membranous
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glucose transporter type 4 (GLUT4) protein contents, GS-E3D restored GLUT4 protein expression to
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the levels of regular diet fed mice, and dose-dependently translocated them to the plasma membrane.
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Conclusion: The results collectively show that GS-E3D ameliorates obesity-related impaired glucose
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tolerance by improving insulin sensitivity in the epidydimal adipose tissue.
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Keywords: GS-E3D; high fat diet; hyperglycemia; insulin resistance; red ginseng
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2
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Abbreviations:
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AMPK, AMP-activated protein kinase; AUC, area under the curve; EWAT, epididymal white adipose
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tissue; FFA, free fatty acid; GLUT4, glucose transporter type 4; H&E, hematoxylin and eosin; HFD,
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high fat diet; HOMA-IR, homeostatic model assessment of insulin resistance; OGTT, oral glucose
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tolerance test; RD, regular diet; RT, room temperature; SEM, standard error of the mean; T2D, type 2
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diabetes
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1. Introduction
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Diabetes mellitus is one of the rapidly growing health concerns worldwide. Diabetes is a
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metabolic disease characterized by aberrant glucose homeostasis and hyperglycemia primarily due to
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impaired insulin action (American Diabetes Association, 2014). It is widely accepted that poor
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glycemic control is a significant risk factor in the pathogenesis of diabetic complications (Fasil et al.,
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2019). Indeed, deleterious consequences on health issues of the world population are estimated if no
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effective clinical interventions for hyperglycemia are provided (Cho et al., 2018). Several studies have
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indicated that lifestyle intervention is clearly effective for the treatment of impaired glucose tolerance
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in diabetes (Tuomilehto et al., 2001; Knowler et al., 2002). Hence it is hard to maintain strict exercise
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and dietary regimens over a long period, patients with impaired glucose tolerance who have not yet
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been prescribed with antidiabetic agents often seek complementary and alternative medicines, mainly
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in the form of dietary supplements or herbal medicines (Pandey et al., 2011). Numerous medicinal
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herbs are recognized to exhibit hypoglycemic effects, thus a better glycemic control can be expected
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in a patient taking herbal medicines (Choudhury et al., 2018).
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Panax ginseng C. A. Mey. is a traditional herbal medicine and its root has been in use for various
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diseases, especially for diabetic treatment (Xie et al., 2005). Now the beneficial effect of ginseng on
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diabetes is corroborated by pre-clinical studies as well as by clinical studies in diabetic individuals
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(Vuksan et al., 2008; Yuan et al., 2012). Traditionally, fresh ginseng is turned into white or red
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ginseng for medicinal use (Nam, 2005). Red ginseng is a processed root of ginseng prepared by
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several cycles of steaming and drying (Lee et al., 2015). Studies on the different methods of
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traditional ginseng processing identified that the major active ingredients of ginseng, ginsenosides, are
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largely dependent on the manufacturing process of ginseng (Nam, 2005; Zheng et al., 2017). Thus,
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diverse studies on processing has been carried out in an effort to maximize medicinal efficacy of
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ginseng extract (Kim et al., 2013; Lee et al., 2016). Likewise, fermentation and enzyme processing on
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ginseng are well reported to increase ginsenoside contents and enhance bioavailability of ginseng
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extract (Choi et al., 2014; Ryu et al., 2013; Sunwoo et al., 2013). Recently, red ginseng preparation by
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enzymatic biotransformation with microbial pectin lyase has been developed and called GS-E3D (Lee
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et al., 2014). GS-E3D has been shown to exhibit inhibitory effects on diseases related to diabetes
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including obesity, hepatic steatosis, formation of advanced glycation end product, diabetic renal
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dysfunction and diabetic retinopathy (Jung et al., 2019; Kim et al., 2017b, 2017a; Lee et al., 2014).
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However, effectiveness of GS-E3D on glycemic control has not yet been studied. Thus, in the present
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study, we aimed to evaluate the preventive effects of GS-E3D on high fat diet-induced glucose
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intolerance in mice.
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2. Materials and methods
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2.1 GS-E3D preparation
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GS-E3D was kindly supplied by the International Ginseng and Herb Research Institute after a
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preparation according to the pervious report (Lee et al., 2014). Briefly, 4-year-old dried Panax
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ginseng C. A. Mey. was purchased from a local market (Wooshin Industrial Co. Ltd., Geumsan,
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Korea) and the specimen is stored at the International Ginseng and Herb Research Institute (No.
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GS201104). Red ginseng extract adjusted to 5 Brix were incubated with 10% pectin lyase (EC
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4.2.2.10, Novozyme, #33095, Denmark) at 50 °C for 5 days in a shaking incubator. To terminate the
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reaction, processed extracts were heated at 95 °C for 10 min, and then dried for further experiment.
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The dried GS-E3D consisted of 62.39 mg/g crude saponin containing the following ginsenosides:
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15.45 mg/g Rb1, 9.48 mg/g Rb2, 9.92 mg/g Rc, 15.58 mg/g Rd, 6.41 mg/g Re, 2.24 mg/g Rf, and 3.33
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mg/g Rg1.
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2.2 Animal experiment
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C57BL/6J male mice (5-week-old) were purchased from Envigo (Indianapolis, IN, USA). Mice
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were fed either regular diet (RD) with 10% kcal% fat (#D12450B) or HFD with 60% kcal% fat
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(#D12492, Research Diets Inc., New Brunswick, NJ, USA) ad libitum and provided free access to
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water in a temperature (22 ± 2°C) and humidity (50 ± 5%) controlled setting with a 12 h light/dark
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cycle. Mice were randomly divided into six groups as follows (n = 8 or 9 per group): a RD-fed group,
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a HFD-fed group, a HFD plus GS-E3D treatment groups (250, 500 or 1000 mg/kg), and a positive
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control group fed HFD plus metformin (250 mg/kg). GS-E3D was dissolved in water and orally
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administered to mice once a day for 6 weeks. RD or HFD-fed control groups received equal volume
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of vehicle during the experiment. After 6 weeks of GS-E3D administration, the mice were fasted
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overnight and sacrificed for further analysis. Epididymal white adipose tissue (EWAT) was dissected
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from mice under ether anesthesia. Dissected tissues were weighed then fixed in the fixatives for
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histological analysis or snap frozen in liquid nitrogen followed by storage at -75 °C for later use. All
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procedures were approved by the Institutional Animal Ethics Committee (IACUC) at Kyung Hee
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University under approval number KHUASP(SE)-16-097.
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2.3 Oral glucose tolerance test
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The oral glucose tolerance tests (OGTT) was performed at week 5 of the experiment. Mice were
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fasted for 6 h and glucose (1.5 g/kg) was orally administered. Prior to glucose administration, blood
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was collected using capillary from tail vein, and repeated 15, 30, 60 and 120 min after administration.
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Blood levels of glucose and insulin were determined by Accu-Chek glucometer (Roche Diagnostics,
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Berlin, Germany) and insulin ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan),
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respectively.
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2.4 Biochemical analysis
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Whole blood was collected by cardiac puncture during animal sacrifice under anesthesia. The
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blood was allowed to clot at room temperature (RT), then centrifuged at 2,000 g for 10 min at 4 °C to
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prepare serum. The serum concentrations of glucose and triglyceride were measured using
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commercial kits (Stanbio Laboratory, Boerne, TX, USA) with an automatic analyzer (SMARTLAB,
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Mannheim, Germany). Level of insulin was determined using Insulin ELISA Kit (Morinaga Institute
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of Biological Science, Yokohama, Japan). Free fatty acids (FFA) in serum were measured by an
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enzymatic colorimetric assay kit (ab65341; Abcam, Cambridge, UK) following the manufacturer’s
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protocol. The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated by
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fasting insulin concentration (µU/ml) x fasting glucose level (mg/dl)/405.
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2.5 Histological analysis
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EWAT were collected from mice under anesthesia for histological analysis. Tissues were fixed in
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10% NBF then embedded in paraffin wax. Tissues in paraffin block were sectioned at 5 µm using
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microtome. Sections were de-paraffinized with xylene and dehydrated by a series of alcohol washes.
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For histological analysis, sectioned EWATs were stained with hematoxylin and eosin (H&E) and
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examined under light microscope (BX51, Olympus, Tokyo, Japan). Images were taken using
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Olympus DP22 digital camera (Tokyo, Japan) and analyzed using Adiposoft as described previously
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(Galarraga et al., 2012).
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2.6 Subcellular fractionation of adipocytes
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Membranes of adipocytes were fractionated as described previously (NISHIUMI and ASHIDA,
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2007). Briefly, EWATs were washed, diced and homogenized in ice-cold fractionation buffer (50 mM
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Tris; pH 8.0, 0.5 mM dithiothreitol, protease inhibitor cocktail and phosphatase inhibitor cocktail)
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containing 0.1 % NP-40. The homogenate was centrifuged at 1,000 g for 10 min at 4 °C. The
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precipitate was suspended in fractionation buffer without NP-40, and re-centrifuged at 1,000 g for 10
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min at 4 °C. The pellet was resuspended in 1.0% NP-40, then centrifuged at 16,000 g for 20 min at
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4 °C to yield the plasma membranes. The supernatant from the first and second centrifugation was
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gathered and centrifuged again at 16,000 g for 20 min at 4 °C, then the supernatant was used as a
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cytosolic fraction. For cell lysate, EWAT was homogenized with ice-cold lysis buffer (10 mM Tris;
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pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and
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0.5 mM dithiothreitol, protease inhibitor cocktail and phosphatase inhibitor cocktail) and centrifuged
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at 16,000 g for 20 min at 4 °C. The supernatant was collected and used as a cell lysate. All reagents
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used were from Sigma-Aldrich (St. Louis, MO, USA).
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2.7 Western blot analysis
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The protein content was quantified using Bio-Rad protein assay kit (Hercules, CA, USA). Equal
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amounts of protein (20 µg/lane) were loaded onto 10% SDS-polyacrylamide gel and electrophoresed
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at constant voltage of 100 V. Proteins were transferred to PVDF membranes (Millipore, Beverly, MA,
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USA). Membranes were blocked with tris-buffered saline with Tween 20 (TBST) containing 5% skim
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milk for 1 h at RT and incubated with anti-glucose transporter type 4 (GLUT4) antibody (1:1000,
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Santa Cruz Biotechnology, Santa Cruz, CA, USA) and beta-actin (1:5000, Abcam) overnight at 4°C.
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The membranes were washed several times, and then incubated with appropriate horseradish
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peroxidase-conjugated secondary antibodies (1:5000, Bethyl Laboratories Inc., Montgomery, TX,
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USA) for 2 h at RT. Protein signal was detected using an enhanced chemiluminescence solution
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(Thermo Scientific, Rockford, IL, USA), and visualized using ImageQuant LAS-4000 (Fugifilm Life
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Science, Tokyo, Japan). The immunoreactive bands in captured images were quantified using ImageJ
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software (http://rsb.info.nih.gov/ij/).
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2.8 Statistical analysis
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All data are expressed as mean ± standard error of the mean (SEM). The results were statistically
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analyzed and compared by using one-way ANOVA followed by Dunnett’s post-test (p < 0.05) in
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Prism 5.01 (GraphPad software, La Jolla, CA, USA).
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3. Results
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3.1 Effects of GS-E3D on weight gain
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Previously, it has been reported that GS-E3D has beneficial effects on obesity (Lee et al., 2014).
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Thus we first examined whether GS-E3D ameliorates HFD-induced obesity. When the changes in
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body weight was monitored during the experimental period, the changes between RD and HFD
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control groups became significant after 2 weeks of HFD feeding (Fig. 1A). Mice treated with E3D-
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1000 showed significant reduction in their body weight after 4 weeks of treatment compared to the
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HFD control group (Fig. 1A). Total weight gains during 6 weeks of experimental period in RD and
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HFD control groups were 4.4 ± 0.2 g and 13.3 ± 1.0 g, respectively (Fig. 1B). The HFD-induced
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weight gain was dose-dependently prevented by GS-E3D treatment in mice. In detail, body weight
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gain was reduced by 16.7%, 20.7% and 26.3% in GS-E3D 250, 500 and 1000 mg/kg treated groups,
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respectively, compared to the HFD control group (Fig. 1B). Consistent with a reduction in body
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weight, EWAT mass was also decreased by 7.3%, 8.6% and 17.1% when normalized for body weight
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in E3D-250, E3D-500 and E3D-1000 groups, respectively, compared with the HFD control group
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(Fig. 1C). Despite the decreased body weight gain in GS-E3D treated groups, there was no significant
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difference in food intake between the HFD control group and GS-E3D treated groups (Fig. 1D).
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3.2 Effects of GS-E3D on metabolic parameters
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Blood concentration of glucose was measured weekly and was compared between groups. As
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shown in Fig. 2A, glucose levels in RD fed mice were stable while a continuous increase in glucose
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levels were observed for mice fed with the HFD. The HFD-induced progression of hyperglycemia
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was slowed down in GS-E3D treated mice and the difference in blood glucose levels was distinct after
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4 weeks of treatment (Fig. 2A). When the serum glucose levels were measured at the end of the
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experiment, all GS-E3D treatment groups showed significantly decreased glucose levels to similar
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degree compared to the HFD control group (E3D-250; 26.4%, E3D-500; 28.1%, E3D-1000; 28.6%;
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Fig. 2B). Likewise, serum insulin levels of GS-E3D treated groups were decreased in a dose
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dependent manner when compared to the HFD control group (Fig. 2C). Having decreased glucose and
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insulin levels, HOMA-IR indices of the mice treated with E3D-250, E3D-500 and E3D-1000 were
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significantly decreased by 41.8%, 45.0% and 55.1%, respectively, compared to that of the HFD
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control mice (Fig. 2D). In addition, serum lipid analysis showed that triglyceride and FFA levels of
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HFD fed mice were elevated by 65.4% and 69.6%, respectively, when compared to those in RD fed
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mice (Fig. 2E and F). GS-E3D treated groups, however, showed reduced levels of triglyceride and
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FFA (26.5% and 33.4% inhibition in E3D-1000 treated group) in a dose dependent manner compared
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to those in HFD control group.
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3.3 Effects of GS-E3D on impaired glucose tolerance
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To determine the effect of GS-E3D on glucose tolerance, OGTT was performed after 5 weeks of
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GS-E3D treatment. Glucose challenge in HFD-fed mice after 6 h fasting dramatically increased the
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blood glucose level compared to RD-fed mice. On the other hand, GS-E3D treated groups had lower
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basal glucose levels and prevented the blood glucose levels from rising in response to glucose load
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(Fig. 3A). The peak blood glucose level, at the 15 min time point, was 30.0% higher in HFD fed mice
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than the RD fed mice while mice treated with E3D-1000 showed 8.2% rise in blood glucose level
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compared to RD fed mice (Fig. 3A). Two hours after glucose administration, blood levels of glucose
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in GS-E3D treated mice were decreased towards basal level whereas blood glucose levels in the HFD
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control mice remained elevated (173.0 ± 7.9 mg/dl in E3D-1000 vs. 243.3 ± 8.3 mg/dl in HFD; Fig.
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3A). HFD-induced rise in glucose area under the curve (AUC) was decreased in mice treated with
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GS-E3D by 19.8% in E3D-1000 treated group compared to the HFD control group (Fig. 3B). To
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assess insulin response after glucose administration, serum insulin levels were measured at different
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time points during OGTT. Insulin levels in HFD fed mice were markedly increased by 4.5-fold after
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15 min of glucose load compared to RD fed mice, indicating development of insulin resistance (Fig.
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3C). In contrast, serum insulin levels in the GS-E3D groups were significantly lower, both fasting and
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after the glucose load, as shown by the insulin AUC. The insulin AUC of the HFD control group was
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considerably increased versus the RD control group while GS-E3D treatment dose-dependently
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reduced insulin AUC by 33.8%, 35.0% and 48.7% in E3D-250, E3D-500 and E3D-1000 treated
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groups, respectively, compared to that of the HFD control group (Fig. 3D).
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3.4 Effects of GS-E3D on adipocyte hypertrophy and GLUT4 translocation
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Adipocyte hypertrophy is closely related to obesity and the development of insulin resistance.
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Thus, histological analysis was performed to compare morphological changes of EWAT between
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experimental groups. The HFD control group exhibited enlarged adipocytes compared to the RD
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control group, while GS-E3D treated groups showed reduced adipocyte size when compared to the
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HFD control group (Fig. 4A). When adipocyte areas were quantified, adipocytes of GS-E3D treated
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mice were distributed over smaller size ranges compared to HFD control mice (Fig. 4B). In addition,
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mean sizes of E3D-250, E3D-500 and E3D-1000 treated adipocytes were 28.2%, 38.5% and 48.7%
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smaller than those of HFD control mice (Fig. 4C).
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stimulated glucose transporter protein GLUT4 in adipocytes as a marker of peripheral insulin
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sensitivity. As shown in Fig. 4D, the protein expression of GLUT4 in EWAT was significantly
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decreased in the HFD control group. Total protein level of GLUT4 was decreased by 28.5% in
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adipocytes of HFD fed mice compared with RD fed mice, however, it was restored with GS-E3D
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treatment in a dose dependent manner (Fig. 4E). Moreover, low levels of the GLUT4 protein in the
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plasma membranes in the HFD control group were reversed in GS-E3D treated groups (35.3%, 60.7%
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and 79.9% increase in E3D-250, E3D-500 and E3D-1000, respectively), demonstrating that GS-E3D
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increases GLUT4 translocation to the plasma membrane (Fig. 4F).
Next, we examined levels of the insulin-
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4. Discussion
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Red ginseng is a traditional herbal medicine used for diabetes, and the effectiveness of red
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ginseng in the maintenance of glycemic control and insulin resistance has been confirmed by several
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human studies (Bang et al., 2014; Vuksan et al., 2008). Recently, an enzymatic modification with
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pectin lyase has been introduced to red ginseng extract in order to increase bioavailability of the
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extract (Lee et al., 2014). This product called GS-E3D has been reported to have therapeutic
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potentials for diabetic complications including nephropathy and retinopathy (Jung et al., 2019; Kim et
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al., 2017a). Sustained hyperglycemia and poor glycemic control are major risk factors for developing
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diabetic complications, and strict glycemic control could prevent or delay onset of chronic
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complications in diabetic patients (ADVANCE Collaborative Group et al., 2008; Fasil et al., 2019;
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Ohkubo et al., 1995). Therefore, it is much of value to investigate whether GS-E3D also exerts anti-
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hyperglycemic activity. Our results in diet-induced obese mouse model confirmed that GS-E3D
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effectively ameliorates metabolic alterations in diabetes including hyperglycemia, hyperinsulinemia,
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hyperlipidemia and insulin resistance.
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Decreased insulin sensitivity play a key role in the pathogenesis of type 2 diabetes (T2D) and the
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OGTT is a widely used procedure to evaluate insulin resistance (Stumvoll et al., 2000). When
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therapeutic potential of GS-E3D in IR was assessed by OGTT, a comparatively low glucose spike and
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reduced AUC of glucose were observed in GS-E3D treatment groups at all doses, suggesting that GS-
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E3D could prevent HFD-fed mice from developing impaired glucose tolerance (Fig. 3B). Moreover, a
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much less stimulation of insulin secretion in response to glucose administration was observed in GS-
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E3D treated mice as demonstrated by decreased insulin AUC (Fig. 3D). The inverse correlation of
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insulin response during an OGTT and peripheral insulin sensitivity has been defined (Abdul-Ghani et
277
al., 2006), and our data indicate that GS-E3D effectively enhances insulin sensitivity. On the contrary,
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Kim et al. reported that GS-E3D did not have any significant effect on blood glucose level in
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streptozotocin (STZ)-induced diabetic rat model (Kim et al., 2017a). STZ induces diabetes in rodents
280
by damaging pancreatic β cells, hence STZ-induced diabetic model is characterized by insulin
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deficiency and hyperglycemia (Eleazu et al., 2013). Our results, along with the findings of Kim et al.
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could imply that GS-E3D can benefit glucose tolerance by improving insulin sensitivity rather than by
283
affecting insulin secretion.
284
Enlargement of adipocytes alongside the accumulation of fat deposition is a potential mediator of
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obesity-related insulin resistance while being a major feature of obesity (Kim et al., 2015). GS-E3D
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has been shown to suppress adipocyte differentiation in vitro and prevent mice from HFD-induced
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weight gain (Lee et al., 2014). In agreement with previous results, GS-E3D significantly reduced
288
weight gain and EWAT mass in mice fed a HFD without changes in food intake (Fig. 1), and these
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effects of GS-E3D were accompanied by inhibition of adipocyte hypertrophy (Fig. 4A). Obese
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hypertrophic adipocytes fail to adequately respond to insulin and consequently FFA levels are
291
increased due to dysregulated lipolysis, which in turn leads to insulin resistance by contributing to
292
peripheral lipotoxicity (Guilherme et al., 2008). High flux of FFAs from adipose tissue transported
293
into the liver, and converted to triglyceride favoring development of fatty liver and
294
hypertriglyceridemia (Qureshi and Abrams, 2007). Based on these facts, our results in which GS-E3D
295
prevented HFD-induced increases in serum triglyceride and FFA levels, despite the lower insulin
296
level, indicate an improvement in insulin sensitivity with GS-E3D (Fig. 2).
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Preventive effect of GS-E3D on insulin resistance was further supported by up-regulation of
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GLUT4 expression in EWAT of HFD-fed mice (Fig. 4D). Adipose tissue is an insulin sensitive organ
299
which contributes to whole-body energy homeostasis mainly via regulation of GLUT4-mediated
300
glucose uptake (Shepherd and Kahn, 1999). The pathogenic association of adipose tissue GLUT4 and
301
insulin resistance has been confirmed in transgenic mice models. Adipocyte-specific knockdown of
302
GLUT4 resulted in insulin resistance whereas overexpression of GLUT4 in adipocytes improved
303
whole-body glucose tolerance (Abel et al., 2001; Carvalho et al., 2005). Our data show a significant
304
increase in cellular GLUT4 expression as well as its translocation to plasma membrane by GS-E3D
305
(Fig. 4). Accordingly, maintenance of insulin sensitivity with GS-E3D in HFD fed mice is probably
306
ascribed to enhancement in peripheral glucose uptake by GS-E3D. It is noteworthy that we did not
307
observe GLUT4 up-regulation by metformin, a positive control drug used in this study. Metformin
308
has been reported to regulate GLUT4 translocation in adipocytes through AMP-activated protein
309
kinase (AMPK) (Lee et al., 2012), although several conflicting studies have reported that metformin
310
as well as AMPK activation have no effect on GLUT4 in adipose tissue of subjects with T2D (Boyle
311
et al., 2011; Virtanen et al., 2003). Several studies proposed that glycemic regulation of ginseng and
312
ginsenosides are attributed to their effect on stimulation of AMPK (Jeong et al., 2014). Whether
313
AMPK induces membrane translocation of GLUT4 in adipocytes remains controversial and thus the
314
detailed mechanism for the up-regulation of GLUT4 translocation by GS-E3D requires further
315
investigation.
316 317
5. Conclusion
318
One of the major value of plant-derived drugs are that they provide synergistic multiple effects.
319
Likewise, our findings suggest that GS-E3D simultaneously exerts anti-hyperglycemic, anti-
320
hyperinsulinemic, anti-hypertriglyceridemic, FFA-lowering as well as weight reducing effects and
321
these multiple activities of GS-E3D may lead to favorable outcomes in individuals with impaired
322
glucose tolerance. The present study indicates that GS-E3D improves insulin sensitivity and glucose
323
tolerance in a mouse model of T2D, which probably attributed increased glucose uptake, suggesting
13
324
that GS-E3D may be useful in clinical practice for the effective prevention or management of insulin
325
resistance and glucose intolerance.
326 327 328
Conflicts of interest The authors declare no competing interest.
329 330 331 332
Author Contributions KGW designed and carried out the experiments, and wrote the manuscript. PMK prepared GSE3D and revised the manuscript. CSH supervised the work and revised the manuscript.
333 334
Acknowledgements
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This research was supported by Korea Institute of Planning and Evaluation for Technology in
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Food, Agriculture, Forestry and Fisheries (IPET) through Export Promotion Technology
337
Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (315049-05-2-
338
SB010) of South Korea.
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Figure legends
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Fig. 1. Effects of GS-E3D on body weight and fat mass in HFD-fed mice. (A) Changes in body
342
weight with or without GS-E3D during the experimental period. (B) Total weight gain during the
343
experiment was calculated. (C) Daily food intake was measured. (D) Weight of epididymal adipose
344
tissue was normalized by total body weight. Data are presented as a mean ± SEM. ## p < 0.01, ### p <
345
0.001 vs. RD; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD.
346 347
Fig. 2. Effects of GS-E3D on glucose tolerance in HFD-fed mice. Blood glucose (A) and serum
348
insulin (C) levels were determined after oral glucose challenge (1.5 g/kg) after 6 h of food deprivation.
349
Area under the curve of blood glucose (B) and insulin (D) were analyzed. AUC, area under curve.
14
350
Data are presented as a mean ± SEM. ### p < 0.001 vs. RD; * p < 0.05, ** p < 0.01, *** p < 0.001 vs.
351
HFD.
352 353
Fig. 3. Effects of GS-E3D on metabolic parameters in HFD-fed mice. (A) Changes in fasting glucose
354
levels with or without GS-E3D. (B) Fasting glucose level and (C) fasting insulin level were measured
355
in the serum. (D) HOMA-IR index was calculated using fasting glucose and fasting insulin levels. (E)
356
Serum levels of triglyceride and (F) free fatty acid were determined. HOMA-IR, homeostatic model
357
assessment for insulin resistance; FFA, free fatty acid. Data are presented as a mean ± SEM.
358
0.001 vs. RD; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD.
###
p<
359 360
Fig. 4. Effects of GS-E3D on adipocyte hypertrophy and GLUT4 translocation in HFD-fed mice. (A)
361
Representative adipose tissue staining by hematoxylin and eosin. Scale bars indicate 100 um. (B)
362
Adipocyte size distribution and (C) adipocyte mean area were measured. (D) Representative western
363
blot images for GLUT4 expression. (E) Quantitative analysis for total protein levels of GLUT4 and (F)
364
GLUT4 translocation in HFD-fed mice. All values were expressed relative to that of the HFD control
365
mice. GLUT4, glucose transporter 4; m-GLUT4, membranous GLUT4; pm-GLUT4, post-
366
membranous GLUT4; t-GLUT4, total-GLUT4. Data are presented as a mean ± SEM. * p < 0.05, ** p
367
< 0.01, *** p < 0.001 vs. HFD.
368 369
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19
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S0378-8741(19)32743-6
DOI:
https://doi.org/10.1016/j.jep.2019.112384
Reference:
JEP 112384
To appear in:
Journal of Ethnopharmacology
Received Date: 9 July 2019 Revised Date:
31 October 2019
Accepted Date: 10 November 2019
Please cite this article as: Kim, G.W., Pyo, M.-K., Chung, S.H., Pectin lyase-modified red ginseng extract improves glucose homeostasis in high fat diet-fed mice, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112384. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
1
Pectin lyase-modified red ginseng extract improves glucose homeostasis in
2
high fat diet-fed mice
3 Go Woon Kim1, Mi-Kyung Pyo2, Sung Hyun Chung1*
4 5 1
6
Department of Pharmacology, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro,
7
Seoul 02447, Republic of Korea 2
8
International Ginseng and Herb Research Institute, Geumsan, Republic of Korea
9 10 11 12
Emails of authors:
13
Kim, Go Woon: [email protected];
14
Pyo, Mi-Kyung: [email protected];
15
Chung, Sung Hyun: [email protected]
16 17 18 19
*
20
Department of Pharmacology, Kyung Hee University, 26 Kyungheedae-ro, Seoul 02447, Republic of
21
Korea
22
Tel: +82-2-961-0373
23
E-mail: [email protected]
Corresponding author: Sung Hyun Chung, Ph.D.
24
1
25
Abstract
26
Ethnopharmacological relevance: Red ginseng has long been used as a traditional folk medicine for
27
various diseases including diabetes. Recently, a preparation of red ginseng extract by pectin lyase
28
modification has been developed and named as GS-E3D.
29
Aim of the study: The aim of this study is to evaluate the preventive effect of GS-E3D on
30
hyperglycemia induced by feeding a high fat diet (HFD) in mice.
31
Materials and Methods: GS-E3D was orally administered to C57BL/6J mice at different doses (250,
32
500, or 1000 mg/kg/day) for 6 weeks while on a HFD. Body weight and blood glucose were
33
monitored weekly, and oral glucose tolerance test (OGTT) was performed at 5th week of the
34
experiment. Glycemic indications and metabolic parameters were further measured in serum.
35
Results: Six weeks of GS-E3D treatment to mice significantly inhibited HFD-induced body weight
36
gain, hyperglycemia, hyperinsulinemia and hypertriglyceridemia. Notably, GS-E3D treated mice at
37
doses of 250, 500 and 1000 mg/kg showed 41.8%, 45.0% and 55.1% reduction in insulin resistance
38
index, respectively, compared to HFD control mice. OGTT revealed that GS-E3D markedly prevented
39
steep rise of blood glucose and insulin levels after glucose challenge and ameliorated HFD-induced
40
glucose and insulin intolerance. The histological analysis showed enlarged adipocytes in HFD-fed
41
mice whereas the adipocyte hypertrophy was prevented in GS-E3D treated mice in a dose-dependent
42
manner. Furthermore, when peripheral glucose uptake level was assessed by total and membranous
43
glucose transporter type 4 (GLUT4) protein contents, GS-E3D restored GLUT4 protein expression to
44
the levels of regular diet fed mice, and dose-dependently translocated them to the plasma membrane.
45
Conclusion: The results collectively show that GS-E3D ameliorates obesity-related impaired glucose
46
tolerance by improving insulin sensitivity in the epidydimal adipose tissue.
47 48
Keywords: GS-E3D; high fat diet; hyperglycemia; insulin resistance; red ginseng
49
2
50
Abbreviations:
51
AMPK, AMP-activated protein kinase; AUC, area under the curve; EWAT, epididymal white adipose
52
tissue; FFA, free fatty acid; GLUT4, glucose transporter type 4; H&E, hematoxylin and eosin; HFD,
53
high fat diet; HOMA-IR, homeostatic model assessment of insulin resistance; OGTT, oral glucose
54
tolerance test; RD, regular diet; RT, room temperature; SEM, standard error of the mean; T2D, type 2
55
diabetes
3
56
1. Introduction
57
Diabetes mellitus is one of the rapidly growing health concerns worldwide. Diabetes is a
58
metabolic disease characterized by aberrant glucose homeostasis and hyperglycemia primarily due to
59
impaired insulin action (American Diabetes Association, 2014). It is widely accepted that poor
60
glycemic control is a significant risk factor in the pathogenesis of diabetic complications (Fasil et al.,
61
2019). Indeed, deleterious consequences on health issues of the world population are estimated if no
62
effective clinical interventions for hyperglycemia are provided (Cho et al., 2018). Several studies have
63
indicated that lifestyle intervention is clearly effective for the treatment of impaired glucose tolerance
64
in diabetes (Tuomilehto et al., 2001; Knowler et al., 2002). Hence it is hard to maintain strict exercise
65
and dietary regimens over a long period, patients with impaired glucose tolerance who have not yet
66
been prescribed with antidiabetic agents often seek complementary and alternative medicines, mainly
67
in the form of dietary supplements or herbal medicines (Pandey et al., 2011). Numerous medicinal
68
herbs are recognized to exhibit hypoglycemic effects, thus a better glycemic control can be expected
69
in a patient taking herbal medicines (Choudhury et al., 2018).
70
Panax ginseng C. A. Mey. is a traditional herbal medicine and its root has been in use for various
71
diseases, especially for diabetic treatment (Xie et al., 2005). Now the beneficial effect of ginseng on
72
diabetes is corroborated by pre-clinical studies as well as by clinical studies in diabetic individuals
73
(Vuksan et al., 2008; Yuan et al., 2012). Traditionally, fresh ginseng is turned into white or red
74
ginseng for medicinal use (Nam, 2005). Red ginseng is a processed root of ginseng prepared by
75
several cycles of steaming and drying (Lee et al., 2015). Studies on the different methods of
76
traditional ginseng processing identified that the major active ingredients of ginseng, ginsenosides, are
77
largely dependent on the manufacturing process of ginseng (Nam, 2005; Zheng et al., 2017). Thus,
78
diverse studies on processing has been carried out in an effort to maximize medicinal efficacy of
79
ginseng extract (Kim et al., 2013; Lee et al., 2016). Likewise, fermentation and enzyme processing on
80
ginseng are well reported to increase ginsenoside contents and enhance bioavailability of ginseng
81
extract (Choi et al., 2014; Ryu et al., 2013; Sunwoo et al., 2013). Recently, red ginseng preparation by
82
enzymatic biotransformation with microbial pectin lyase has been developed and called GS-E3D (Lee
4
83
et al., 2014). GS-E3D has been shown to exhibit inhibitory effects on diseases related to diabetes
84
including obesity, hepatic steatosis, formation of advanced glycation end product, diabetic renal
85
dysfunction and diabetic retinopathy (Jung et al., 2019; Kim et al., 2017b, 2017a; Lee et al., 2014).
86
However, effectiveness of GS-E3D on glycemic control has not yet been studied. Thus, in the present
87
study, we aimed to evaluate the preventive effects of GS-E3D on high fat diet-induced glucose
88
intolerance in mice.
89 90
2. Materials and methods
91
2.1 GS-E3D preparation
92
GS-E3D was kindly supplied by the International Ginseng and Herb Research Institute after a
93
preparation according to the pervious report (Lee et al., 2014). Briefly, 4-year-old dried Panax
94
ginseng C. A. Mey. was purchased from a local market (Wooshin Industrial Co. Ltd., Geumsan,
95
Korea) and the specimen is stored at the International Ginseng and Herb Research Institute (No.
96
GS201104). Red ginseng extract adjusted to 5 Brix were incubated with 10% pectin lyase (EC
97
4.2.2.10, Novozyme, #33095, Denmark) at 50 °C for 5 days in a shaking incubator. To terminate the
98
reaction, processed extracts were heated at 95 °C for 10 min, and then dried for further experiment.
99
The dried GS-E3D consisted of 62.39 mg/g crude saponin containing the following ginsenosides:
100
15.45 mg/g Rb1, 9.48 mg/g Rb2, 9.92 mg/g Rc, 15.58 mg/g Rd, 6.41 mg/g Re, 2.24 mg/g Rf, and 3.33
101
mg/g Rg1.
102 103
2.2 Animal experiment
104
C57BL/6J male mice (5-week-old) were purchased from Envigo (Indianapolis, IN, USA). Mice
105
were fed either regular diet (RD) with 10% kcal% fat (#D12450B) or HFD with 60% kcal% fat
106
(#D12492, Research Diets Inc., New Brunswick, NJ, USA) ad libitum and provided free access to
107
water in a temperature (22 ± 2°C) and humidity (50 ± 5%) controlled setting with a 12 h light/dark
108
cycle. Mice were randomly divided into six groups as follows (n = 8 or 9 per group): a RD-fed group,
109
a HFD-fed group, a HFD plus GS-E3D treatment groups (250, 500 or 1000 mg/kg), and a positive
5
110
control group fed HFD plus metformin (250 mg/kg). GS-E3D was dissolved in water and orally
111
administered to mice once a day for 6 weeks. RD or HFD-fed control groups received equal volume
112
of vehicle during the experiment. After 6 weeks of GS-E3D administration, the mice were fasted
113
overnight and sacrificed for further analysis. Epididymal white adipose tissue (EWAT) was dissected
114
from mice under ether anesthesia. Dissected tissues were weighed then fixed in the fixatives for
115
histological analysis or snap frozen in liquid nitrogen followed by storage at -75 °C for later use. All
116
procedures were approved by the Institutional Animal Ethics Committee (IACUC) at Kyung Hee
117
University under approval number KHUASP(SE)-16-097.
118 119
2.3 Oral glucose tolerance test
120
The oral glucose tolerance tests (OGTT) was performed at week 5 of the experiment. Mice were
121
fasted for 6 h and glucose (1.5 g/kg) was orally administered. Prior to glucose administration, blood
122
was collected using capillary from tail vein, and repeated 15, 30, 60 and 120 min after administration.
123
Blood levels of glucose and insulin were determined by Accu-Chek glucometer (Roche Diagnostics,
124
Berlin, Germany) and insulin ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan),
125
respectively.
126 127
2.4 Biochemical analysis
128
Whole blood was collected by cardiac puncture during animal sacrifice under anesthesia. The
129
blood was allowed to clot at room temperature (RT), then centrifuged at 2,000 g for 10 min at 4 °C to
130
prepare serum. The serum concentrations of glucose and triglyceride were measured using
131
commercial kits (Stanbio Laboratory, Boerne, TX, USA) with an automatic analyzer (SMARTLAB,
132
Mannheim, Germany). Level of insulin was determined using Insulin ELISA Kit (Morinaga Institute
133
of Biological Science, Yokohama, Japan). Free fatty acids (FFA) in serum were measured by an
134
enzymatic colorimetric assay kit (ab65341; Abcam, Cambridge, UK) following the manufacturer’s
135
protocol. The homeostatic model assessment for insulin resistance (HOMA-IR) was calculated by
136
fasting insulin concentration (µU/ml) x fasting glucose level (mg/dl)/405.
6
137 138
2.5 Histological analysis
139
EWAT were collected from mice under anesthesia for histological analysis. Tissues were fixed in
140
10% NBF then embedded in paraffin wax. Tissues in paraffin block were sectioned at 5 µm using
141
microtome. Sections were de-paraffinized with xylene and dehydrated by a series of alcohol washes.
142
For histological analysis, sectioned EWATs were stained with hematoxylin and eosin (H&E) and
143
examined under light microscope (BX51, Olympus, Tokyo, Japan). Images were taken using
144
Olympus DP22 digital camera (Tokyo, Japan) and analyzed using Adiposoft as described previously
145
(Galarraga et al., 2012).
146 147
2.6 Subcellular fractionation of adipocytes
148
Membranes of adipocytes were fractionated as described previously (NISHIUMI and ASHIDA,
149
2007). Briefly, EWATs were washed, diced and homogenized in ice-cold fractionation buffer (50 mM
150
Tris; pH 8.0, 0.5 mM dithiothreitol, protease inhibitor cocktail and phosphatase inhibitor cocktail)
151
containing 0.1 % NP-40. The homogenate was centrifuged at 1,000 g for 10 min at 4 °C. The
152
precipitate was suspended in fractionation buffer without NP-40, and re-centrifuged at 1,000 g for 10
153
min at 4 °C. The pellet was resuspended in 1.0% NP-40, then centrifuged at 16,000 g for 20 min at
154
4 °C to yield the plasma membranes. The supernatant from the first and second centrifugation was
155
gathered and centrifuged again at 16,000 g for 20 min at 4 °C, then the supernatant was used as a
156
cytosolic fraction. For cell lysate, EWAT was homogenized with ice-cold lysis buffer (10 mM Tris;
157
pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and
158
0.5 mM dithiothreitol, protease inhibitor cocktail and phosphatase inhibitor cocktail) and centrifuged
159
at 16,000 g for 20 min at 4 °C. The supernatant was collected and used as a cell lysate. All reagents
160
used were from Sigma-Aldrich (St. Louis, MO, USA).
161 162
2.7 Western blot analysis
7
163
The protein content was quantified using Bio-Rad protein assay kit (Hercules, CA, USA). Equal
164
amounts of protein (20 µg/lane) were loaded onto 10% SDS-polyacrylamide gel and electrophoresed
165
at constant voltage of 100 V. Proteins were transferred to PVDF membranes (Millipore, Beverly, MA,
166
USA). Membranes were blocked with tris-buffered saline with Tween 20 (TBST) containing 5% skim
167
milk for 1 h at RT and incubated with anti-glucose transporter type 4 (GLUT4) antibody (1:1000,
168
Santa Cruz Biotechnology, Santa Cruz, CA, USA) and beta-actin (1:5000, Abcam) overnight at 4°C.
169
The membranes were washed several times, and then incubated with appropriate horseradish
170
peroxidase-conjugated secondary antibodies (1:5000, Bethyl Laboratories Inc., Montgomery, TX,
171
USA) for 2 h at RT. Protein signal was detected using an enhanced chemiluminescence solution
172
(Thermo Scientific, Rockford, IL, USA), and visualized using ImageQuant LAS-4000 (Fugifilm Life
173
Science, Tokyo, Japan). The immunoreactive bands in captured images were quantified using ImageJ
174
software (http://rsb.info.nih.gov/ij/).
175 176
2.8 Statistical analysis
177
All data are expressed as mean ± standard error of the mean (SEM). The results were statistically
178
analyzed and compared by using one-way ANOVA followed by Dunnett’s post-test (p < 0.05) in
179
Prism 5.01 (GraphPad software, La Jolla, CA, USA).
180 181
3. Results
182
3.1 Effects of GS-E3D on weight gain
183
Previously, it has been reported that GS-E3D has beneficial effects on obesity (Lee et al., 2014).
184
Thus we first examined whether GS-E3D ameliorates HFD-induced obesity. When the changes in
185
body weight was monitored during the experimental period, the changes between RD and HFD
186
control groups became significant after 2 weeks of HFD feeding (Fig. 1A). Mice treated with E3D-
187
1000 showed significant reduction in their body weight after 4 weeks of treatment compared to the
188
HFD control group (Fig. 1A). Total weight gains during 6 weeks of experimental period in RD and
189
HFD control groups were 4.4 ± 0.2 g and 13.3 ± 1.0 g, respectively (Fig. 1B). The HFD-induced
8
190
weight gain was dose-dependently prevented by GS-E3D treatment in mice. In detail, body weight
191
gain was reduced by 16.7%, 20.7% and 26.3% in GS-E3D 250, 500 and 1000 mg/kg treated groups,
192
respectively, compared to the HFD control group (Fig. 1B). Consistent with a reduction in body
193
weight, EWAT mass was also decreased by 7.3%, 8.6% and 17.1% when normalized for body weight
194
in E3D-250, E3D-500 and E3D-1000 groups, respectively, compared with the HFD control group
195
(Fig. 1C). Despite the decreased body weight gain in GS-E3D treated groups, there was no significant
196
difference in food intake between the HFD control group and GS-E3D treated groups (Fig. 1D).
197 198
3.2 Effects of GS-E3D on metabolic parameters
199
Blood concentration of glucose was measured weekly and was compared between groups. As
200
shown in Fig. 2A, glucose levels in RD fed mice were stable while a continuous increase in glucose
201
levels were observed for mice fed with the HFD. The HFD-induced progression of hyperglycemia
202
was slowed down in GS-E3D treated mice and the difference in blood glucose levels was distinct after
203
4 weeks of treatment (Fig. 2A). When the serum glucose levels were measured at the end of the
204
experiment, all GS-E3D treatment groups showed significantly decreased glucose levels to similar
205
degree compared to the HFD control group (E3D-250; 26.4%, E3D-500; 28.1%, E3D-1000; 28.6%;
206
Fig. 2B). Likewise, serum insulin levels of GS-E3D treated groups were decreased in a dose
207
dependent manner when compared to the HFD control group (Fig. 2C). Having decreased glucose and
208
insulin levels, HOMA-IR indices of the mice treated with E3D-250, E3D-500 and E3D-1000 were
209
significantly decreased by 41.8%, 45.0% and 55.1%, respectively, compared to that of the HFD
210
control mice (Fig. 2D). In addition, serum lipid analysis showed that triglyceride and FFA levels of
211
HFD fed mice were elevated by 65.4% and 69.6%, respectively, when compared to those in RD fed
212
mice (Fig. 2E and F). GS-E3D treated groups, however, showed reduced levels of triglyceride and
213
FFA (26.5% and 33.4% inhibition in E3D-1000 treated group) in a dose dependent manner compared
214
to those in HFD control group.
215 216
3.3 Effects of GS-E3D on impaired glucose tolerance
9
217
To determine the effect of GS-E3D on glucose tolerance, OGTT was performed after 5 weeks of
218
GS-E3D treatment. Glucose challenge in HFD-fed mice after 6 h fasting dramatically increased the
219
blood glucose level compared to RD-fed mice. On the other hand, GS-E3D treated groups had lower
220
basal glucose levels and prevented the blood glucose levels from rising in response to glucose load
221
(Fig. 3A). The peak blood glucose level, at the 15 min time point, was 30.0% higher in HFD fed mice
222
than the RD fed mice while mice treated with E3D-1000 showed 8.2% rise in blood glucose level
223
compared to RD fed mice (Fig. 3A). Two hours after glucose administration, blood levels of glucose
224
in GS-E3D treated mice were decreased towards basal level whereas blood glucose levels in the HFD
225
control mice remained elevated (173.0 ± 7.9 mg/dl in E3D-1000 vs. 243.3 ± 8.3 mg/dl in HFD; Fig.
226
3A). HFD-induced rise in glucose area under the curve (AUC) was decreased in mice treated with
227
GS-E3D by 19.8% in E3D-1000 treated group compared to the HFD control group (Fig. 3B). To
228
assess insulin response after glucose administration, serum insulin levels were measured at different
229
time points during OGTT. Insulin levels in HFD fed mice were markedly increased by 4.5-fold after
230
15 min of glucose load compared to RD fed mice, indicating development of insulin resistance (Fig.
231
3C). In contrast, serum insulin levels in the GS-E3D groups were significantly lower, both fasting and
232
after the glucose load, as shown by the insulin AUC. The insulin AUC of the HFD control group was
233
considerably increased versus the RD control group while GS-E3D treatment dose-dependently
234
reduced insulin AUC by 33.8%, 35.0% and 48.7% in E3D-250, E3D-500 and E3D-1000 treated
235
groups, respectively, compared to that of the HFD control group (Fig. 3D).
236 237
3.4 Effects of GS-E3D on adipocyte hypertrophy and GLUT4 translocation
238
Adipocyte hypertrophy is closely related to obesity and the development of insulin resistance.
239
Thus, histological analysis was performed to compare morphological changes of EWAT between
240
experimental groups. The HFD control group exhibited enlarged adipocytes compared to the RD
241
control group, while GS-E3D treated groups showed reduced adipocyte size when compared to the
242
HFD control group (Fig. 4A). When adipocyte areas were quantified, adipocytes of GS-E3D treated
243
mice were distributed over smaller size ranges compared to HFD control mice (Fig. 4B). In addition,
10
244
mean sizes of E3D-250, E3D-500 and E3D-1000 treated adipocytes were 28.2%, 38.5% and 48.7%
245
smaller than those of HFD control mice (Fig. 4C).
246
stimulated glucose transporter protein GLUT4 in adipocytes as a marker of peripheral insulin
247
sensitivity. As shown in Fig. 4D, the protein expression of GLUT4 in EWAT was significantly
248
decreased in the HFD control group. Total protein level of GLUT4 was decreased by 28.5% in
249
adipocytes of HFD fed mice compared with RD fed mice, however, it was restored with GS-E3D
250
treatment in a dose dependent manner (Fig. 4E). Moreover, low levels of the GLUT4 protein in the
251
plasma membranes in the HFD control group were reversed in GS-E3D treated groups (35.3%, 60.7%
252
and 79.9% increase in E3D-250, E3D-500 and E3D-1000, respectively), demonstrating that GS-E3D
253
increases GLUT4 translocation to the plasma membrane (Fig. 4F).
Next, we examined levels of the insulin-
254 255
4. Discussion
256
Red ginseng is a traditional herbal medicine used for diabetes, and the effectiveness of red
257
ginseng in the maintenance of glycemic control and insulin resistance has been confirmed by several
258
human studies (Bang et al., 2014; Vuksan et al., 2008). Recently, an enzymatic modification with
259
pectin lyase has been introduced to red ginseng extract in order to increase bioavailability of the
260
extract (Lee et al., 2014). This product called GS-E3D has been reported to have therapeutic
261
potentials for diabetic complications including nephropathy and retinopathy (Jung et al., 2019; Kim et
262
al., 2017a). Sustained hyperglycemia and poor glycemic control are major risk factors for developing
263
diabetic complications, and strict glycemic control could prevent or delay onset of chronic
264
complications in diabetic patients (ADVANCE Collaborative Group et al., 2008; Fasil et al., 2019;
265
Ohkubo et al., 1995). Therefore, it is much of value to investigate whether GS-E3D also exerts anti-
266
hyperglycemic activity. Our results in diet-induced obese mouse model confirmed that GS-E3D
267
effectively ameliorates metabolic alterations in diabetes including hyperglycemia, hyperinsulinemia,
268
hyperlipidemia and insulin resistance.
269
Decreased insulin sensitivity play a key role in the pathogenesis of type 2 diabetes (T2D) and the
270
OGTT is a widely used procedure to evaluate insulin resistance (Stumvoll et al., 2000). When
11
271
therapeutic potential of GS-E3D in IR was assessed by OGTT, a comparatively low glucose spike and
272
reduced AUC of glucose were observed in GS-E3D treatment groups at all doses, suggesting that GS-
273
E3D could prevent HFD-fed mice from developing impaired glucose tolerance (Fig. 3B). Moreover, a
274
much less stimulation of insulin secretion in response to glucose administration was observed in GS-
275
E3D treated mice as demonstrated by decreased insulin AUC (Fig. 3D). The inverse correlation of
276
insulin response during an OGTT and peripheral insulin sensitivity has been defined (Abdul-Ghani et
277
al., 2006), and our data indicate that GS-E3D effectively enhances insulin sensitivity. On the contrary,
278
Kim et al. reported that GS-E3D did not have any significant effect on blood glucose level in
279
streptozotocin (STZ)-induced diabetic rat model (Kim et al., 2017a). STZ induces diabetes in rodents
280
by damaging pancreatic β cells, hence STZ-induced diabetic model is characterized by insulin
281
deficiency and hyperglycemia (Eleazu et al., 2013). Our results, along with the findings of Kim et al.
282
could imply that GS-E3D can benefit glucose tolerance by improving insulin sensitivity rather than by
283
affecting insulin secretion.
284
Enlargement of adipocytes alongside the accumulation of fat deposition is a potential mediator of
285
obesity-related insulin resistance while being a major feature of obesity (Kim et al., 2015). GS-E3D
286
has been shown to suppress adipocyte differentiation in vitro and prevent mice from HFD-induced
287
weight gain (Lee et al., 2014). In agreement with previous results, GS-E3D significantly reduced
288
weight gain and EWAT mass in mice fed a HFD without changes in food intake (Fig. 1), and these
289
effects of GS-E3D were accompanied by inhibition of adipocyte hypertrophy (Fig. 4A). Obese
290
hypertrophic adipocytes fail to adequately respond to insulin and consequently FFA levels are
291
increased due to dysregulated lipolysis, which in turn leads to insulin resistance by contributing to
292
peripheral lipotoxicity (Guilherme et al., 2008). High flux of FFAs from adipose tissue transported
293
into the liver, and converted to triglyceride favoring development of fatty liver and
294
hypertriglyceridemia (Qureshi and Abrams, 2007). Based on these facts, our results in which GS-E3D
295
prevented HFD-induced increases in serum triglyceride and FFA levels, despite the lower insulin
296
level, indicate an improvement in insulin sensitivity with GS-E3D (Fig. 2).
12
297
Preventive effect of GS-E3D on insulin resistance was further supported by up-regulation of
298
GLUT4 expression in EWAT of HFD-fed mice (Fig. 4D). Adipose tissue is an insulin sensitive organ
299
which contributes to whole-body energy homeostasis mainly via regulation of GLUT4-mediated
300
glucose uptake (Shepherd and Kahn, 1999). The pathogenic association of adipose tissue GLUT4 and
301
insulin resistance has been confirmed in transgenic mice models. Adipocyte-specific knockdown of
302
GLUT4 resulted in insulin resistance whereas overexpression of GLUT4 in adipocytes improved
303
whole-body glucose tolerance (Abel et al., 2001; Carvalho et al., 2005). Our data show a significant
304
increase in cellular GLUT4 expression as well as its translocation to plasma membrane by GS-E3D
305
(Fig. 4). Accordingly, maintenance of insulin sensitivity with GS-E3D in HFD fed mice is probably
306
ascribed to enhancement in peripheral glucose uptake by GS-E3D. It is noteworthy that we did not
307
observe GLUT4 up-regulation by metformin, a positive control drug used in this study. Metformin
308
has been reported to regulate GLUT4 translocation in adipocytes through AMP-activated protein
309
kinase (AMPK) (Lee et al., 2012), although several conflicting studies have reported that metformin
310
as well as AMPK activation have no effect on GLUT4 in adipose tissue of subjects with T2D (Boyle
311
et al., 2011; Virtanen et al., 2003). Several studies proposed that glycemic regulation of ginseng and
312
ginsenosides are attributed to their effect on stimulation of AMPK (Jeong et al., 2014). Whether
313
AMPK induces membrane translocation of GLUT4 in adipocytes remains controversial and thus the
314
detailed mechanism for the up-regulation of GLUT4 translocation by GS-E3D requires further
315
investigation.
316 317
5. Conclusion
318
One of the major value of plant-derived drugs are that they provide synergistic multiple effects.
319
Likewise, our findings suggest that GS-E3D simultaneously exerts anti-hyperglycemic, anti-
320
hyperinsulinemic, anti-hypertriglyceridemic, FFA-lowering as well as weight reducing effects and
321
these multiple activities of GS-E3D may lead to favorable outcomes in individuals with impaired
322
glucose tolerance. The present study indicates that GS-E3D improves insulin sensitivity and glucose
323
tolerance in a mouse model of T2D, which probably attributed increased glucose uptake, suggesting
13
324
that GS-E3D may be useful in clinical practice for the effective prevention or management of insulin
325
resistance and glucose intolerance.
326 327 328
Conflicts of interest The authors declare no competing interest.
329 330 331 332
Author Contributions KGW designed and carried out the experiments, and wrote the manuscript. PMK prepared GSE3D and revised the manuscript. CSH supervised the work and revised the manuscript.
333 334
Acknowledgements
335
This research was supported by Korea Institute of Planning and Evaluation for Technology in
336
Food, Agriculture, Forestry and Fisheries (IPET) through Export Promotion Technology
337
Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (315049-05-2-
338
SB010) of South Korea.
339 340
Figure legends
341
Fig. 1. Effects of GS-E3D on body weight and fat mass in HFD-fed mice. (A) Changes in body
342
weight with or without GS-E3D during the experimental period. (B) Total weight gain during the
343
experiment was calculated. (C) Daily food intake was measured. (D) Weight of epididymal adipose
344
tissue was normalized by total body weight. Data are presented as a mean ± SEM. ## p < 0.01, ### p <
345
0.001 vs. RD; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD.
346 347
Fig. 2. Effects of GS-E3D on glucose tolerance in HFD-fed mice. Blood glucose (A) and serum
348
insulin (C) levels were determined after oral glucose challenge (1.5 g/kg) after 6 h of food deprivation.
349
Area under the curve of blood glucose (B) and insulin (D) were analyzed. AUC, area under curve.
14
350
Data are presented as a mean ± SEM. ### p < 0.001 vs. RD; * p < 0.05, ** p < 0.01, *** p < 0.001 vs.
351
HFD.
352 353
Fig. 3. Effects of GS-E3D on metabolic parameters in HFD-fed mice. (A) Changes in fasting glucose
354
levels with or without GS-E3D. (B) Fasting glucose level and (C) fasting insulin level were measured
355
in the serum. (D) HOMA-IR index was calculated using fasting glucose and fasting insulin levels. (E)
356
Serum levels of triglyceride and (F) free fatty acid were determined. HOMA-IR, homeostatic model
357
assessment for insulin resistance; FFA, free fatty acid. Data are presented as a mean ± SEM.
358
0.001 vs. RD; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HFD.
###
p<
359 360
Fig. 4. Effects of GS-E3D on adipocyte hypertrophy and GLUT4 translocation in HFD-fed mice. (A)
361
Representative adipose tissue staining by hematoxylin and eosin. Scale bars indicate 100 um. (B)
362
Adipocyte size distribution and (C) adipocyte mean area were measured. (D) Representative western
363
blot images for GLUT4 expression. (E) Quantitative analysis for total protein levels of GLUT4 and (F)
364
GLUT4 translocation in HFD-fed mice. All values were expressed relative to that of the HFD control
365
mice. GLUT4, glucose transporter 4; m-GLUT4, membranous GLUT4; pm-GLUT4, post-
366
membranous GLUT4; t-GLUT4, total-GLUT4. Data are presented as a mean ± SEM. * p < 0.05, ** p
367
< 0.01, *** p < 0.001 vs. HFD.
368 369
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19
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