Ginsenoside Re Ameliorates Brain Insulin Resistance and Cognitive

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Ginsenoside Re Ameliorates Brain Insulin Resistance and Cognitive Dysfunction in High-fat Diet-induced C57BL/6 Mice Jong Min Kim, Chang Hyeon Park, Seon Kyeong Park, Tae Wan Seung, Jin Yong Kang, Jeong Su Ha, Du Sang Lee, Uk Lee, Dae Ok Kim, and Ho Jin Heo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00297 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017

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Journal of Agricultural and Food Chemistry

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Running Title: Ginsenoside Re attenuated cognitive impairment in C57BL/6 mice

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Ginsenoside Re Ameliorates Brain Insulin Resistance and

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Cognitive Dysfunction in High-fat Diet-induced C57BL/6 Mice

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☆ ☆ Jong Min Kim†,☆ , Chang Hyeon Park†,☆ , Seon Kyeong Park†, Tae Wan Seung†,

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Jin Yong Kang†, Jeong Su Ha†, Du Sang Lee†, Uk Lee§, Dae-Ok Kim#, Ho Jin Heo†,*

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-----------------------------------------------------------------------------------------------------------------------------------

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*

Corresponding author at: Division of Applied Life Science(BK21 plus), Institute of

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Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea.

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Tel: +82 55 772 1907; Fax: +82 55 772 1909. E-mail address: [email protected] (H. J. Heo)

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Author detail

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†

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Gyeongsang National University, Jinju 52825, Republic of Korea

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§

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Republic of Korea

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#

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Republic of Korea

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CHP: [email protected], JMK: [email protected], SKP: [email protected],

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TWS: [email protected], JYK: [email protected], JSH: [email protected],

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DSL: [email protected], UL: [email protected], DOK: [email protected], HJH:

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[email protected]

Division of Applied Life Science (BK21 plus), Institute of Agriculture and Life Science,

Division of Special Purpose Trees, National Institute of Forest Science, Suwon 16631,

Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104,

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☆

These authors contributed equally to this work as the co-first author. 1

ACS Paragon Plus Environment


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ABSTRACT

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The ameliorating effects of ginsenoside Re (G Re) on high-fat diet (HFD)-induced insulin

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resistance in C57BL/6 mice were investigated to assess its physiological function. In the

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results of behavioral tests, G Re improved cognitive dysfunction on diabetic mice using Y-

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maze, passive avoidance and Morris water maze tests. G Re also significantly recovered

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hyperglycemia and fasting blood glucose level. In the results of serum analysis, G Re

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decreased triglyceride (TG), total cholesterol (TCHO), low density lipoprotein cholesterol

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(LDLC), glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase

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(GPT), and increased the ratio of high density lipoprotein cholesterol (HDLC). G Re

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regulated the acetylcholine (ACh), acetylcholinesterase (AChE), malondialdehyde (MDA),

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superoxide dismutase (SOD) and oxidized glutathione (GSH)/total GSH by regulating the c-

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Jun N-terminal protein kinase (JNK) pathway. These findings suggest that G Re could be

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used to improve HFD-induced insulin resistance condition by ameliorating hyperglycemia via

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protecting the cholinergic and antioxidant system in the mice brains.

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Keywords: Cognitive impairment, diabetes mellitus, high-fat diet, ginsenoside Re, JNK

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pathway

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INTRODUCTION

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Diabetes mellitus (DM) is a chronic disease abnormally progressed by

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hyperglycemia, and also, may damage almost all organs in the body, such as the central and

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peripheral nerves, retinas, liver, and kidneys. Recently, there have been increasing issues

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associated with DM and its complications, in particular, diabetes-associated cognitive

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impairment (DACM).1 Although the DM-induced mechanisms of cognitive deficiency are

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not fully understood, hyperglycemia and insulin resistance seem to be markedly associated

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with DM. In addition, it has been consistently suggested that oxidative stress is connected

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with the progression of diabetic complications.2 Oxidative stresses also play an important role

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in DACM. Simultaneously, chronic consumption of a high-fat diet (HFD) may cause the

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production of reactive oxygen species (ROS) and mitochondrial impairment.3 The damaged

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mitochondrial function in a neuronal cell leads to a deficit of insulin signaling, which may

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trigger insulin resistance and hyperinsulinemia. Recent studies have considered the role of

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insulin resistance in the cognitive impairment and pathological progress of Alzheimer’s

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disease (AD).4 Insulin resistance is also associated with phosphorylated c-Jun N-terminal

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kinase (p-JNK), which leads to phosphorylation of a serine site in the insulin receptor

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substrate (IRS), and continuously impedes serine/threonine kinase (Akt) signaling.5

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Consequently, inhibited Akt signaling is caused by decrease of phosphorylated Akt (p-Akt),

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the activated form, and induce the activation of GSK-3β, which expedites the

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phosphorylation of tau attached in microtubules and aggregation of phosphorylated tau (p-

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tau).6 Moreover, an abnormal insulin signal and activated GSK-3β precipitate

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neurodegeneration promoting the formation of neurofibrillary tangles (NFT), leading to

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neuronal death.7 Finally, in neurons, p-JNK can trigger the impairment of energy production

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processes and cause neuronal inflammation, cognitive dysfunction and signaling associated 3



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with AD.5 Also, p-JNK up-regulates the expression of Bax and Bad as pro-apoptosis factors,

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and impedes the Bcl-2 as survival factor. An increase in these pro-apoptotic factors stimulates

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cytochrome c release from the mitochondria to cytosol, and consistently, expedites the

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activation and cleavage of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP)

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related with apoptosis in the neuronal cell.8

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Ginsenoside Re (PubChem CID: 441921, G Re) is a triterpenoid saponin compound

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typically isolated from Panax ginseng. Recently, our research showed that the extract of

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Panax ginseng berry had an anti-amnesic effect, and G Re was identified as the main

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compound by ultra-performance liquid chromatography/quadrupole time of flight tandem

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mass spectrometry (Q-TOF UPLC/MS2).9 G Re improves cognitive impairment against

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scopolamine, NaNO2 and ethanol-induced oxidative stress.10 G Re also has an anti-diabetic

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effect including the protection of the kidney and eyes against diabetic oxidative stress, and

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remarkably ameliorates DACM in streptozotocin-induced type 1 diabetic rats.11-12

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Nevertheless, there is relatively limited information on the cognitive ability associated with

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type 2 diabetes mellitus (T2DM) induced by the HFD. For this reason, this study aimed to

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evaluate whether G Re can improve HFD-induced T2DM associated cognitive dysfunction,

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using various in vivo tests, ex vivo biochemical changes and the JNK pathway.

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MATERIALS AND METHODS

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Materials. Ginsenoside Re was purchased from Aktin Chemicals, Inc. (Chengdu,

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China).

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metaphosphoric acid, 4-vinylpyridine, bovine serum albumin (BSA), glucose, dimethyl

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sulfoxide (DMSO), hydrochloride, iron (Ш) chloride hexahydrate, sodium hydroxide,

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superoxide dismutase (SOD) determination kit, and all other chemicals used in this study

Acetylthiocholine

iodide,

thiobarbituric

acid

4


(TBA),

phosphoric

acid,

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were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The total

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glutathione (GSH) kit was purchased from Enzo Life science Inc. (Farmingdale, NY, USA).

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Animals and treatment. C57BL/6 mice (4 weeks-old, male) were purchased from

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supplier (Samtako, Osan, Korea). Mice were housed 3 per cage, and freely allowed access to

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water and feed, and controlled in standard laboratory conditions (steady temperature, 23±1°C;

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humidity, 55±5% and 12 h light/dark cycle). All experiments followed the guidelines

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produced by the Animal Care and Use Committee of Gyeongsang National University

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(certificate: GNU-131105-M0067). All mice excluding the control group were fed with the

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HFD for 8 weeks. After induction of high glucose status on mice, the G Re was mixed in

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purified water (5, 10 and 20 mg/kg of body weight; G Re 5, G Re 10 and G Re 20,

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respectively), and administered intragastrically once per day for 4 weeks. Daily food intake

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and weekly body weight were measured. The overall design of the experiment is shown in

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Figure 1.

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Behavioral tests for cognitive evaluation. Y-maze is constituted as triple opaquely

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white plastic arms. Each mouse which was located at the end of designated arm is allowed to

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freely explore the maze for 8 min. The sequences of arm entries were assessed using a video

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system (Smart 3.0 video tracking system, Panlab, Barcelona, Spain). The ratio of spatial

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perception was measured as the ratio of the number of entries.13

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Passive avoidance test chamber is separated to a light compartment and a dark

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compartment, with a gate between the two compartments. When the mice entered the dark

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zone, an electrical shock was provide to entered mice (0.5 mA, 3 sec). Mice will learn to

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associate certain properties of the chamber with the foot shock. Next day, the mice were put

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into non-dark area, and the latency times until entering to the dark compartment were 5



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measured for 5 min.14

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Morris water maze consists of a round pool divided into quadrant (N, S, E and W

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zones, respectively), and each quadrant zones was indicated to different visual cues. This

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pool was filled with opaque water using skim milk, depth of water was 30 cm, and

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temperature was maintained at 20±2°C. The platform was fixed into the steady location in the

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W zone during training session (day 1-4). In the training test, mice freely were swam until

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they reached the platform to escape from the opaque water during maximum 60 sec. Latency

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time to arrive in the hidden platform was recorded in each training trial using the video

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system (Panlab). After training test, the probe test was conducted. In the probe test, the

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platform was removed from the test zone, and mice allowed to freely swimming for 90 sec.15

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Intraperitoneal glucose tolerance test (IPGTT) and measurement of fasting

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blood glucose. To measure the IPGTT, mice were fasted during 4 h. After that, D-glucose (2

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g/kg of body weight) was injected in the abdominal cavity of mice, and blood samples were

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taken at 0, 30, 60, 90, and 120 min after the intraperitoneal injection. Glucose levels in the

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fasting state, which collected from tip of the tail vein, was measured at every week using

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Accu-Chek glucose meter (Roche Diagnostics GmbH, Mannheim, Germany).

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Collection of serum and brain tissues. After behavioral test, mice which had

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undergone fasting for 12 h were sacrificed by CO2 inhalation for biochemical studies. The

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serum was collected from abdominal aorta. The collected brain tissue was homogenized

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using a bullet blender (Nextadvance Inc., Averill Park, NY, USA) with 10-fold volumes of

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phosphate buffered saline (PBS) for acetylcholine (ACh), acetylcholinesterase (AChE),

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malondialdehyde (MDA) and SOD assay, and 20-fold volumes of 5% metaphosphoric acid

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for GSH assay in an ice bath. The concentration of cerebral protein was determined through a

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Bradford protein assay.13 6


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Serum biochemicals. The collected blood sample was stored at room temperature for

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20 min in heparin tube. And then, the supernatant was collected from obtained blood sample

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centrifuged at 10,000×g for 15 min at 4°C, and has conducted serum analysis. The glutamic

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oxaloacetic transaminase (GOT), glutamine pyruvic transaminase (GPT), blood urea nitrogen

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(BUN), creatine (CRE), total cholesterol (TCHO), triglyceride (TG), and high density

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lipoprotein cholesterol (HDLC) concentrations of serum were measured using clinical

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chemistry analyzer (Fuji dri-chem4000i; Fuji film Co., Tokyo, Japan). Low density

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lipoprotein cholesterol (LDLC) was determined as follow; LDLC (mg/dl) = TCHO - (HDLC

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+ TG/5).16

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AChE activity and ACh level in brain tissue. The homogenized brain tissues at the

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PBS were centrifuged to collect the supernatant to measure AChE activity and ACh level,

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respectively at 14,000×g for 30 min at 4°C. AChE activity was determined using the

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colorimetric method.17 The supernatant with 50 mM sodium phosphate buffer (pH 8.0) was

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reacted at 37°C for 15 min. Acetylthiocholine iodide solution (0.5 mM) and buffered

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Ellman's reagent [1 mM 5,50-dithio-bis (2-nitrobenzoic acid) in 50 mM sodium phosphate

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buffer (pH 8.0)] were reacted at room temperature for 10 min. Absorbance was measured at

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405 nm. ACh level was estimated as described by the colorimetric method.18 The supernatant

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was added to alkaline hydroxylamine reagent [2 M hydroxylamine in HCl and 3.5 N sodium

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hydroxide] at room temperature for 1 min. Then, 0.5 N HCl and 0.37 M FeCl3 in 0.1N HCl

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(pH 1.2) was added. The absorbance measured at 540 nm.

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MDA level, SOD content, and ratio of oxidized GSH/total GSH in brain tissue.

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The MDA levels were implemented in accordance with colorimetric way by combining

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between the MDA in the brain tissues in mice and TBA. The homogenized tissues at the PBS

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were centrifuged at 2,500×g for 10 min at 4°C. After that, the acquired supernatants were 7



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mixed with 1% phosphoric acid and 0.67% TBA in water bath for 1 h at 95°C. And then, this

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reactants were centrifuged at 600×g for 10 min to discard the insoluble substances. This

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supernatants were measured at 532 nm, and the MDA contents were expressed as nmole/mg

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of protein.13

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To measure the SOD content, homogenized brain tissues at the PBS were centrifuged

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at 400×g for 10 min at 4°C, and after these supernatants were removed. 1×Cell Extraction

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Buffer containing the 10% SOD buffer, 0.4% (v/v) Triton X-100, and 200 µM

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phenylmethane sulfonylfluoride in distilled water were added into pellet, and incubated for

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30 min on the ice bath. This reactant was centrifuged at 10,000×g for 10 min at 4°C. The

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supernatant was acquired, and used according to guided protocol of the commercial kit.

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To measure the total GSH and oxidized GSH ratio, the homogenized brain tissues at

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5% metaphosphoric acid were centrifuged at 14,000×g for 15 min at 4°C. And then,

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supernatant and 2 M 4-vinylpyridine were incubated at room temperature for 1 h to measure

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the oxidized GSH. The evaluation of GSH was performed using the commercial kit.

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Western blot analysis. The brain tissues were homogenized for 15 min with

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ProtinExTM Animal cell/tissue (GeneAll Biotechnology, Seoul, Korea) with 1% protease

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inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). After that, homogenates

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were centrifuged at 13,000×g for 10 min at 4oC, and these supernatants were collected and

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stored at -80oC. The supernatant was loaded into sodium dodecyl sulfate polyacrylamide

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(SDS-PAGE) gel electrophoresis, and electro-transferred to a poly-vinylidene difluoride

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(PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were reacted in tris-

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buffered saline (TBS) containing 0.1% Tween 20 (TBST) containing 1:4000 diluted solutions

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of mouse monoclonal primary antibody to bind β-actin, p-JNK (Thr183/Tyr185), p-tau

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(Ser404), and cleaved PARP (Asp214; c-PARP) or 1:4000 diluted solutions of rabbit 8


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monoclonal primary antibody to combine phosphorylated IRS (Ser307; p-IRS) at 4oC. After

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that, membrane was reacted with horserdish peroxide-conjugated anti-mouse IgG (1:2000) or

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anti-rabbit IgG (1:5000) secondary antibody solution for 1 h in TBST including 5% skim

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milk. For chemiluminescence detection, the immune complexes were incubated by

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chemiluminescence with ECL reagent (Bionote, Hwaseong, Korea). The density of band was

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measured by Image J software (National Institutes of Health, Bethesda, MD, USA).

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Statistical analysis. All data was presented as the mean±standard deviation (SD).

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The statistical significance of differences between the groups was measured by a one-way

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analysis of variance (ANOVA). The significance of differences was conducted by the

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Duncan's new multiple-range test (p‹0.05) of SAS ver. 9.1 (SAS Institute Inc., Cary, NC,

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USA).

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RESULTS

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Body weight and food intake. To evaluate the ameliorating effect of G Re on HFD-

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induced weight regulation, the weight gain, and food intake values were measured (Table 1.).

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The final body weight of the control, HFD, and G Re 5, 10, and 20 groups showed 33.25 g,

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50.50 g, 48.25 g, 47.33 g, and 45.23 g, respectively. The food intake of the control, HFD, and

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G Re 5, 10 and 20 groups was 2.36 g/day, 3.18 g/day, 2.74 g/day, 2.56 g/day, and 2.46 g/day,

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respectively.

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Blood glucose and insulin tolerance. The fasting blood glucose during the sample

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intake period is presented in Figure 2A. After starting the HFD, the fasting blood glucose

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level of the HFD group was significantly higher than the control group. However, G Re

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administrations improved the abnormal fasting blood glucose level. To confirm the regulation

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of insulin tolerance on HFD-induced T2DM, the intraperitoneal glucose tolerance (IPGT) 9



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was also measured at 0, 15, 30, 60, and 90 min (Figure 2B).

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Before glucose injection (0 min), the groups treated with the HFD (312.47 mg/dL)

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showed a hyperglycemic state compared to the control group (185.00 mg/dL), and all groups

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showed the highest glucose level at 15 min. The glucose level of the control group returned to

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a normal state after 90 min (190.42 mg/dL). In contrast, the HFD group presented a lower

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width of decreased at a faster rate between 15 and 90 min (528.37 mg/dL and 395.11 mg/dL,

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respectively) than the other groups. However, the sample groups (G Re 5, 357.23 mg/dL; G

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Re 10, 320.23 mg/dL; and G Re 20, 281.32 mg/dL, respectively) showed improved glucose

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tolerance compared to the HFD group.

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in vivo behavioral tests. The learning and memory effects against diabetes-

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associated cognitive dysfunction were sequentially evaluated by the Y-maze, passive

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avoidance, and Morris water maze tests (Figure 3A-F). Alternation behavior of the HFD

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group (23.56%) was decreased compared to the control group (52.23%). However, those of

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the sample groups (G Re 5, 41.76%; G Re 10, 44.33%, and G Re 20, 46.89%, respectively)

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were statistically improved (Figure 3A). All groups, except the HFD group, showed a similar

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number of arm entries. However, the HFD group showed only a decreased movement caused

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by HFD-induced obesity in the arms (Figure 3B).

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The HFD group had a significantly shortened latency time before entering the dark

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room in the retention trial (182 .00 sec) compared to that of the control group (300.00 s).

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Whereas, the G Re 10 (300.00 sec) and G Re 20 group (300.00 sec) showed similar

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improvement in short-term memory compared to the HFD group (Figure 3C).

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The escape latency time of the HFD group (51.03 sec at day 1 to 37.12 sec at day 4)

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was relatively higher than the control group (38.15 sec at day 1 to 13.69 sec at day 2).

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However, those of the G Re groups (G Re 5, 44.40 sec to 23.92 sec; G Re 10, 43.38 sec to 10


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21.83 sec, and G Re 20, 39.43 sec to 16.84 sec, respectively) were significantly decreased

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compared to the HFD group (Figure 3D). In the probe test, long-term memory and the

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spontaneous behavioral ability of the HFD group was significantly decreased (37.18% stay

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ratio in the target area) compared to that of the control group (56.18%). However, the G Re

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treatment groups (G Re 5, 40.51%; G Re 10, 45.05%, and G Re 20, 49.95%, respectively)

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showed improved long-term cognitive function (Figure 3E) compared to that of the HFD

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group. In addition, the path tracing showed the mice of the control group circled around the

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area marked with a circle (W zone) on the platform, relatively more than the other groups,

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whereas, the mice of the HFD group circled around all zones (Figure 3F). These tracks made

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by the mice also present learning and memory ability regarding eliminated platforms in the

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area marked with a circle (W zone).

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Serum biochemicals. Effects of G Re on changes of serum biochemicals in the

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HFD-induced mice were analyzed (Table 2). The contents of the GOT and GPT have

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generally been used as hepatotoxic indicators.19 The contents of both indicators in the G Re

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groups (G Re 5, 110.33 U/L and 80.13 U/L; G Re 10, 103.33 U/L and 50.32 U/L, and G Re

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20, 86.32 U/L and 39.24 U/L, respectively) were decreased compared to those of the HFD

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group (130.67 U/L and 110.25 U/L). The levels of the TG, TCHO, HDLC and LDLC are

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commonly used to assess the serum lipid state, such as hyperlipidemia and

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hypercholesterolemia.20 The levels of these lipid indicators, excluding the HDLC in the HFD

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group, was considerably increased compared to that of the control group. In contrast, the

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levels of these indicators in the G Re groups were significantly decreased compared to those

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in the HFD groups, and the increased HDLC level contrasted with the decreased TG, TCHO,

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and LDLC levels.

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AChE activity and ACh level in brain tissue. In the current study, the AChE 11



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activity was increased in the HFD group (110.14%) compared to that of the control group

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(100.00%). However, that of the G Re groups (G Re 5, 102.56%; G Re 10, 98.36%, and G Re,

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91.55%, respectively) was dose-dependently decreased (Figure 4A). The ACh level showed

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the opposite result to the AChE activity (Figure 4B). The ACh content of HFD group (1.77

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mmole/mg of protein) was increased compared to that of the control group (1.99 mmole/mg

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of protein). However, that of the G Re groups were represented as G Re 5, 1.78 mmole/mg of

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protein; G Re 10, 1.89 mmole/mg of protein, and G Re, 2.03 mmole/mg of protein,

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respectively.

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MDA level, SOD content, and oxidized GSH /total GSH ratio. The protective

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effect of G Re against lipid peroxidation was measured by the MDA level (Figure 5A). The

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MDA level of the HFD group (4.51 nmol/mg of protein) increased compared to the control

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group (3.45 nmol/mg protein). However, the sample groups (G Re 5, 4.23 nmol/mg of protein;

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G Re 10, 3.71 nmol/mg of protein, and G Re 20, 3.35 nmol/mg of protein, respectively)

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gradually decreased the MDA level compared to that of the HFD group. The SOD content of

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the HFD group (3.18 U/mg of protein) was decreased in the brain because of the increased

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oxidative stress compared to the control group (4.50 U/mg). However, the sample groups (G

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Re 5, 3.51 U/mg of protein; G Re 10, 4.12 U/mg of protein; and G Re 20, 4.46 U/mg of

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protein, respectively) statistically increased the SOD content by decreasing the oxidative

283

stress compared to that of the HFD group (Figure 5B). In addition, the ratio of oxidized

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GSH/total GSH of the HFD group (50.23%) was drastically increased compared to that of the

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control group (34.25%). In contrast, the administration of G Re (G Re 5, 43.33%; G Re 10,

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39.12%, and G Re 20, 35.13%, respectively) showed a dose-dependent improvement in the

287

ratio of oxidized GSH/total GSH (Figure 5C).

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Cerebral apoptotic signaling. The ameliorating effect of G Re on cerebral insulin 12


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signaling was examined in the brain of the HFD mice via western blot assay (Figure 6). The

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level of p-JNK expression in the brain of the HFD mice was significantly up-regulated

291

compared to the control group (Figure 6B). In contrast, in the G Re treatment groups, it was

292

down-regulated compared to the HFD group. Also, p-IRS and p-tau expression levels in the

293

HFD group were considerably increased compared to the control group, while, in the

294

respective G Re groups the levels were remarkably decreased compared to the HFD group

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(Figure 6C, D). While, c-PARP expression of HFD group is not significant compared to that

296

of control group. However, that of G Re group is considerably reduced compared to that of

297

HFD group (Figure 6E).

298 299

DISCUSSION

300

The two primary forms of DM are known to type 1 and 2 diabetes. Both types are

301

connected with progressive β-cell impairment.21 Type 1 DM (T1DM) results from mainly

302

impairment of pancreatic β-cell due to an autoimmune-mediated impairment.22 And also,

303

these β-cell loss is induced by activated macrophages and T-cells, and cytokines, nitric oxide

304

(NO), and free radicals secreted by these cells23. Whereas, T2DM, one of the most common

305

metabolic disorders, is related to impaired insulin signaling and insulin resistance.21 T2DM is

306

related to dysfunction of carbohydrate, lipid and protein metabolism with serious effects

307

resulting in long-term complications. These complications are associated with the

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physiological abnormalities and problems in organ, and may cause cerebrovascular disease

309

and dementia.4 The causes of these DM are known to excess of glucorticoids and growth

310

hormone, pregnancy, Lipodystrophy, mutations of insulin receptor, and obesity/overweight

311

through high calorie diet.24 Especially, the administration of HFD is associated with the

312

T2DM, obesity, and diverse metabolic syndromes, and HFD can induce the collapse of 13



313

antioxidant system in brain as well as increased hepatocellular TG contents and lipid

314

peroxidation in liver. 1, 2 And, these factors lead to insulin resistance in brain, and ultimately

315

cause the cognitive dysfunction and AD.5 Therefore, we studied whether G Re can ameliorate

316

the HFD-induced metabolic disease related with T2DM such as the insulin resistance, the

317

memory impairment, hyperglycemia, hypercholesterolemia, dysfunction of the antioxidant

318

system and cholinergic system, and impaired metabolic pathway in diabetic mice brain.

319

HFD is closely related to weight gain. When consuming more than a certain amount

320

of calories during long-term intake, excess carbohydrates promote de novo lipogenesis, which

321

promotes fat accumulation, particularly in the liver and adipocyte. And also, because insulin

322

stimulates de novo lipogenesis, when insulin resistance in developed, it can not only

323

gradually increase insulin levels, but also make it easier to accumulate fat. Thus, increased fat

324

in liver and adipocyte will increase insulin resistance, body weight and glucose level in

325

blood.25 To determine the effect of G Re on weight gain, body weight and calorie intake were

326

measured (Table 1). The HFD group showed increased body weight compared with that of

327

control group. However, G Re groups showed reduced body weight than HFD group.

328

Previous

329

phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), fatty acid

330

synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) in HepG2 cells and HFD-induced

331

C57BL/6J mice, and ultimately it inhibited hyperglycemia and hepatic steatosis.26

research reported

G Re regulated

fat accumulation factors including

332

Abnormal fasting glucose and glucose tolerance are associated with the development

333

of insulin resistance and signaling.27 Moreover, insulin resistance negatively influences

334

signaling between the brain, insulin, and cognitive functioning.28 To evaluate the

335

improvement effect of insulin resistance through the consumption of the G Re, blood glucose

336

level during sample intake periods, and IPGTT were conducted (Figure 2). The HFD groups 14


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Page 15 of 41


337

showed hyperglycemia status related with insulin resistance through the long-term

338

administration of the HFD (Figure 1A). However, the G Re groups showed improved blood

339

glucose level through the consumption of the G Re. And also, the G Re groups presented

340

ameliorated insulin resistance compared with the HFD group (Figure 2B). The Panax ginseng

341

berry extract containing the G Re improved the blood glucose level and the insulin

342

resistance.9 Also, these results suggest that G Re administration may have a positive effect on

343

HFD-induced T2DM by regulating the hyperglycemic state and insulin tolerance.

344

The consumption of the G Re considerably improved the HFD-induced cognitive

345

deficit in the Y-maze, passive avoidance and Morris water maze tests (Figure 3). In the Y-

346

maze test, when a mouse is exposed to a new environment, instinctively they prefer to

347

navigate a new environment rather than return to the previously visited arms.29 However,

348

HFD-induced diabetic mouse show impaired structures of hippocampal synaptic plasticity

349

and abnormal neurotransmitter, so they tend to re-enter the previous visited arm.30 The HFD

350

group showed spatial working and behavioral memory deficit through reduced spontaneous

351

alternative behavior and number of arm entries compared with the control group (Figure 3A,

352

B). In contrast, the G Re groups presented ameliorated spatial and behavioral memory

353

function. Short-term learning and memory ability associated with activation of N-methyl-D-

354

aspartic acid (NMDA) receptors and amygdala in the brain tissue were assessed using the

355

passive avoidance test.31 The HFD groups showed lowered steps-through latency in the

356

retention trial compared with that of the control group (Figure 3C). However, the G Re

357

groups presented ameliorated cognitive function similar to that of the control group. The

358

Morris water maze test is based on the reward principle. The Morris water maze test

359

examined the long-term memory and spontaneous behavioral ability. Long-term learning and

360

memory ability is usually trained across four trials, and spontaneous alternation behavior is 15



361

measured by the probe test.15 In this results, the HFD group showed impaired long-term

362

learning and cognitive function compared with the control group (Figure 3D). Escape latency

363

time of all groups is decreased. However, the HFD group showed lower change of escape

364

latency time against that of the control group in the final trail. And also, the retention time in

365

target zone of the HFD group was reduced (Figure 3E, F). Contrastively, G Re groups

366

showed increased escape latency time and retention time in the target zone. Previous research

367

reported that mice treated with HFD for 1-8 mouths might lead to abnormalities of learning

368

and memory functions associated with hippocampal-dependent performance.32 Exclusive

369

oxidative stress appearing in the hyperglycemic state in the cerebral region also leads to nerve

370

damage and cell death.33 Consequently, activation of NMDA receptors was decreased, and

371

continuous deactivation of NMDA receptors could cause the deficit of post-achieving

372

memory and long-term memory.31 A recent study reported that G Re remarkably alleviated

373

the cognitive dysfunction in streptozotocin-induced diabetic mice by decreasing oxidative

374

stress and inflammation.12 Considering these studies, learning and memory abilities could be

375

decreased by HFD-induced insulin resistance and oxidative stress, and G Re may be used to

376

improve DM-associated cognitive defects.

377

Hypercholesteremia, hyperlipidemia, polyphagia and hyperglycemia are typical

378

diabetic features.2 Administration of HFD increases the serum TG and free fatty acid (FFA)

379

levels, expedites adipose development, and subsequently increases the body weight of mice.

380

The fat absorbed from the diet is dissolved by bile acid and permeated into the blood as TGs,

381

and FFAs are cleaved from the TGs by lipoprotein lipase.34 The FFAs are transported into the

382

blood as albumin complexes. This complex is extensively carried to the internal tissues, such

383

as the liver. The accumulated FFA and TG in the liver promote the excessive production of

384

very-low-density lipoprotein (VLDL) cholesterol in the liver and blood circulation.35 16


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Page 17 of 41


385

Previous research showed that G Re-rich Panax ginseng berry particularly G Re, improved

386

the serum lipid state and down-regulated the blood glucose levels, TG, TCHO, and LDLC. In

387

addition, it improved the HDLC levels in HFD-induced mice.36 Our results suggest that the

388

serum lipid balance may be affected by the G Re treatment by reducing the hepatic injury

389

indicators (GOT and GPT), and improving the serum lipid status (TG, TCHO, HDLC, and

390

LDLC) in HFD-induced mice (Table 2), and ultimately, it may have an ameliorating effect on

391

HFD-induced hyperlipidemia in the mice.

392

The cholinergic system is associated with cognitive function. AChE activity and ACh

393

level in the cholinergic neuronal system are also associated with behaviorally sensory

394

signaling events. ACh is separated to acetate and choline by the AChE in the cerebral

395

synaptic cleft.37 AChE is fixed in the lipid bilayer and tends to be altered by lipid

396

peroxidation. ACh is a chemically stable compound which can exist in a stable state for a

397

long time after being secreted into the neuronal synaptic cleft.38 However, AChE from the

398

lipid membrane damaged by lipid peroxidation related factors such as MDA and 4-

399

hydroxynonenal (4-HNE) increases the activity. They accelerate the degradation of ACh,

400

which normally plays a role, and break down into acetic acid and choline.37 Excessive

401

activation of the AChE is associated with abnormal deformation of the cell membrane, and

402

this causes abnormal neurotransmission and cognitive dysfunction.39 An increased AChE

403

activation, which lead to ACh degradation and neurotransmitter disorder was found in

404

diabetic rats.37 Therefore, HFD-induced impaired cholinergic system ultimately induces

405

cognitive impairment originating from synaptic dysfunction. When HFD-induced oxidative

406

stress was excessively generated, AChE activity was increased.37 G Re stimulates the release

407

of neurotransmitters, such as dopamine and ACh, by passing through the BBB, and normally

408

promotes neurotransmission in the neuronal system.40 In this study, G Re groups showed 17



409

reduced activation of AChE and increased ACh content in cerebral tissue compared with that

410

of the HFD group (Figure 4), and G Re seemed to improve the HFD-induced cholinergic

411

defects via inhibition of AChE activity and decrease of ACh decomposition.

412

SOD is an enzyme that catalyzes the partitioning of the superoxide radical to either

413

non-toxic molecular oxygen or hydrogen peroxide.41 Hydrogen peroxide is continuously

414

converted to H2O by glutathione peroxidase (GPx) using cytosolic GSH. When these

415

constituents of antioxidant system are reduced, ROS is not sufficiently scavenged, and it

416

causes oxidative stress, which can induce the lipid peroxidation and cell membrane

417

deformation. And, these stress is known to continuously cause cell death by producing the

418

MDA and numerous free radicals.42 Alteration of the ratio between total GSH and oxidized

419

GSH, the form that scavenges the radicals, is biologically used to assess oxidative cell

420

damage.43 MDA is a secondary metabolite of lipid peroxidation, and it is used as a biomarker

421

of oxidative stress during the onset of numerous diseases.42 Long-term HFD feed generates

422

the ROS in cerebral mitochondria, and impairs the antioxidant system, which can scavenge

423

the these ROS, including SOD and GSH.44 And, Kuhad et al.45 reported that DM increases

424

the MDA and nitrite levels, and also, decreases the GSH levels, SOD contents and catalase

425

activity in the brain of rats. In addition, the reduction of MDA may be associated with the

426

activity of AChE (figure 4A). G Re inhibited lipid peroxidation and ultimately may suppress

427

the activity of AChE by protecting the neuronal lipid bilayer, and reduce the decomposition

428

of ACh. Kim et al. 44 showed that HFD-induced diabetic condition led to impairment of the

429

antioxidant system, and accompanied cognitive dysfunction. However, Panax ginseng berry,

430

including G Re, treatment improved the lipid status, SOD contents, and ratio of oxidized

431

GSH/total GSH by lowering the oxidative stress in HFD-induced mice.9 In this study, G Re

432

groups showed increased SOD contents and reduced oxidized GSH/total GSH ratio and MDA 18


Page 18 of 41

Page 19 of 41


433

content against HFD group (Figure 5). Consequently, our results suggest that the G Re could

434

have an ameliorating effect on HFD-induced cognitive deficits through the inhibition of lipid

435

peroxidation and down-regulation of antioxidant enzymes caused by insulin resistance.

436

Insulin resistance, a typical feature of T2DM, impedes the insulin signal in neuronal

437

cells, and this deficit of cerebral insulin signal and insulin secretion was associated with AD

438

neurodegeneration.24 In the normal status, insulin binds to insulin receptors, thereby

439

regulating the insulin signal, which expedites glucose uptake by the neurons. Also, activation

440

of the insulin receptors leads to phosphorylation of IRS (tyrosine site), which promotes PI3-K

441

phosphorylation and glucose uptake.46 If the insulin signaling is abnormally impaired,

442

however, insulin resistance is developed in most tissues. Particularly, a high-calorie diet

443

induces insulin resistance, and inhibits the reaction of insulin and its receptors. This abnormal

444

signal leads to JNK phosphorylation, which phosphorylates the serine site of IRS instead of

445

the tyrosine site.47 This increased p-IRS with its phosphorylated serine site impedes PI3-K

446

phosphorylation and significantly impedes Akt activation associated with cell survival. The

447

inhibited Akt induces GSK-3β activation, and expedites p-tau aggregation and development

448

of the NFT. This formation of p-tau and NFT ultimately accelerates abnormal structural

449

modification of cell and neuronal death.7 Recent research also showed that the expression of

450

the insulin receptor, p-IRS (tyrosine site), Akt, and PI3-K in the brain was considerably

451

decreased in the cerebral insulin signaling mechanism of diabetes-induced models.48 A

452

number of apoptotic cascades can be induced by the release of cytochrome c from the

453

mitochondria into the cytosol, where it binds to the apoptotic protease activating factor 1

454

(Apaf-1), leading to binding of procaspase-9 and activation of procaspase-9 by proteolytic

455

processes.49 When Akt is inactivated by mediators of oxidative stress, caspase-9 is activated

456

by inducing the leakage of cytochrome c.50 Therefore, the release of cytochrome c to the 19



Page 20 of 41

457

cytosol also activates the caspase pathway and cleaves the PARP. As a result of this, decrease

458

of NAD and ATP was presented in the cerebral tissue of diabetic models.51,

459

stimulates apoptosis through DNA fragmentation and ultimately leads to cell death.51

460

Polyphenolic compounds could down-regulate the dysfunctions of insulin signaling in brain

461

tissue by alleviating the expression of signaling proteins associated with the JNK pathway.44

462

Our research suggests that G Re may improve insulin resistance by regulating the associated

463

protein expression levels, such as p-JNK, p-IRS, p-tau, and c-PARP levels. Finally, G Re may

464

alleviate diabetes associated cognitive dysfunctions in the HFD-induced mice.

52

c-PARP

465

Ginsenoside Re has an ameliorating effect on HFD-induced hyperglycemic C57BL/6

466

mice by improving insulin tolerance, including blood glucose. G Re significantly regulated

467

food intake associated with weight gain and serum lipid statuses, such as TG, TCHO, HDLC,

468

and LDLC levels. The administration of G Re also improved the learning and memory

469

dysfunction in HFD-induced cognitive impairment mice. The anti-amnesic effect of G Re

470

may be produced by cholinergic recovery (AChE inhibition and ACh increase) and protection

471

of antioxidant systems (SOD contents and ratio of oxidized GSH/total GSH) in the brain

472

tissue damaged by HFD-induced oxidative stress. An abnormal insulin signal via the

473

regulation of the JNK pathway in brain tissue was ameliorated by G Re administration.

474

Consequently, the ameliorating effect of G Re on the DACM could be induced by cholinergic

475

recovery, activation of antioxidant systems, and improved insulin tolerance and signaling in

476

brain tissue. Therefore, G Re can be used as a natural potential resource to ameliorate

477

learning and memory dysfunction associated with HFD-induced hyperglycemic status.

478 479

AUTHOR INFORMATION

480

Corresponding author 20


Page 21 of 41

481


*(H.J.H.) Phone: +82 55 772 1907. Fax: +82 55 772 1909. Email: [email protected].

482 483

Funding Information

484

This study was supported by Basic Science Research Program through the National

485

Research Foundation of Korea (NRF-2015R1D1A3A01015931) funded by the Ministry of

486

Education, Republic of Korea. C.H.P., J.M.K., S.K.P., T.W.S., J.Y.K., J.S.H., and D.S.L. were

487

supported by the BK21 Plus program, Ministry of Education, Republic of Korea.

488 489 490

Notes The authors declare no competing financial interest.

491

21



492

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Table 1. Effect of ginsenoside Re on body weight and food intake Group

Control

HFD

G Re 5

G Re 10

G Re 20

Wight gain (g)

33.25±1.25e

50.50±3.32a

48.25±2.22b

47.33±0.57c

45.23±0.41d

Food intake (kcal/day)

9.09±4.43e

16.66±1.15a

14.36±0.84b

13.41±0.73c

12.89±0.94d

Results shown are mean±SD (݊=8). Data were statistically considered at ‫

Ginsenoside Re Ameliorates Brain Insulin Resistance and Cognitive

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