Oleuropein-Rich Diet Attenuates Hyperglycemia and Impaired

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Journal of Agricultural and Food Chemistry

Oleuropein-Rich Diet Attenuates Hyperglycemia and Impaired Glucose Tolerance in Type 2 Diabetes Model Mouse

Kazutoshi Murotomi1∗, Aya Umeno1, Mayu Yasunaga1, Mototada Shichiri2, Noriko Ishida2, Taisuke Koike3, Toshiki Matsuo3, Hiroko Abe1, Yasukazu Yoshida1, Yoshihiro Nakajima1

1

Health Research Institute, National Institute of Advanced Industrial Science and

Technology (AIST), Takamatsu, Kagawa 761-0395, Japan 2

Health Research Institute, National Institute of Advanced Industrial Science and

Technology (AIST), Ikeda, Osaka 563-8577, Japan 3

Eisai Food & Chemical Co., Ltd., Chuo-ku, Tokyo 103-0027, Japan

∗Correspondence: Kazutoshi Murotomi, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan. TEL/FAX: +81-87-869-4207/+81-87-869-4178, E-mail: [email protected]

Short title: Effects of oleuropein-rich diet on type 2 diabetic phenotypes

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ACS Paragon Plus Environment


1

Abstract

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Oleuropein, a phenolic compound found in abundance in olive leaves, has

3

beneficial effects on various diseases. However, it is unknown whether an oleuropein-rich

4

diet is efficacious against type 2 diabetic phenotypes. In this study, we investigated the

5

effects of the oleuropein-containing supplement OPIACE, whose oleuropein content

6

exceeds 35% (w/w), on the diabetic phenotypes in type 2 diabetes model Tsumura Suzuki

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Obese Diabetes (TSOD) mouse. TSOD mice were fed OPIACE at 4 weeks of age, i.e.,

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before the TSOD mice exhibited diabetic phenotypes. We revealed that OPIACE attenuated

9

hyperglycemia and impaired glucose tolerance in TSOD mice over the long term (from 10

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to 24 weeks of age), but had no effect on obesity. Furthermore, we demonstrated that

11

OPIACE mildly reduced oxidative stress in TSOD mice by 26.2%, and attenuated

12

anxiety-like behavioral abnormality in aged TSOD mice. The results suggest that

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oleuropein suppresses the progression of type 2 diabetes and diabetes-related behavioral

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abnormality over the long term.

15 16

Key words: type 2 diabetes, oleuropein, hyperglycemia, impaired glucose tolerance,

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anxiety

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■ Introduction

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Type 2 diabetes mellitus is a chronic and common lifestyle disease that is caused

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by complex interactions between multiple susceptibility genes and environmental factors1.

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The pathogenesis of type 2 diabetes is characterized by the progressive decline in insulin

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action (insulin resistance) in peripheral tissues, accompanied by the impairment of insulin

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secretion by pancreatic β cells to compensate for the insulin resistance, leading to overt

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hyperglycemia1. Although insulin resistance and the impairment of insulin secretion are

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caused by complex and multiple mechanisms, oxidative stress is an early risk factor in the

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pathogenesis of type 2 diabetes2. We have demonstrated that the type 2 diabetes model

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Tsumura Suzuki Obese Diabetes (TSOD) mouse, a polygenic model of obese type 2

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diabetes3, is exposed to a constant level of oxidative stress before and after the onset of

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diabetes4. In addition, insulin resistance and insulin secretory function are exacerbated by

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oxidative stress5. Thus, it is expected that the intake of antioxidants may be an effective

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means of preventing type 2 diabetes.

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Chronic hyperglycemia leads to severe and lethal complications, including

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atherosclerosis, neuropathy, nephropathy, and retinopathy6. It has been reported that

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Alzheimer’s disease7, Parkinson’s disease8, depression9, and anxiety disorder10 are also

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kinds of diabetic complications. The risk of microvascular complications in diabetes

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patients was reduced by improving long-term glycemic control11. It was shown that

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cognitive function in type 2 diabetes patients was negatively associated with hemoglobin

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A1c level12. Thus, the control of blood glucose level is a critical step to prevent the

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progression of type 2 diabetes and its complications.

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In traditional medicine, natural products have been used for the treatment of

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diabetes. It has been reported that various plant components, including triterpenoids13 and

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flavonoids14, exert anti-diabetic effects. Polyphenols are also a kind of plant component that

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is regularly consumed as part of the human diet. Numerous studies have demonstrated that

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polyphenols have beneficial effects on various diseases because of their antioxidant,

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anti-inflammatory,

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Oleuropein (Figure 1), a phenolic compound found in abundance in Olea europaea leaves,

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has several pharmacological activities, including antioxidant, anti-inflammatory, and

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antimicrobial activities16. As oleuropein exhibits potent antioxidant activity in vitro17, it is

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speculated to be the main component responsible for the potentially beneficial effects of

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olive leaves on various diseases18. In fact, previous studies have indicated that oleuropein

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ameliorates hepatic steatosis19, cardiovascular diseases20, and neurodegenerative diseases21.

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In addition, hyperglycemia in type 1 diabetes model animals22 and metabolic abnormalities

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in high-carbohydrate and high-fat diet rats20 were partially attenuated by feeding

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oleuropein-rich extracts. However, it is unknown whether an oleuropein-rich diet is

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efficacious against type 2 diabetic phenotypes, including chronic hyperglycemia and

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impaired glucose tolerance.

antimicrobial,

anticancer,

and

immunomodulatory

activities15.

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In the present study, we investigated the effect of the oleuropein-containing

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supplement OPIACE, an olive leaf extract whose oleuropein content exceeds 35% (w/w),

60

on the diabetic phenotypes in type 2 diabetes model TSOD mice. We found that OPIACE

61

attenuated hyperglycemia and impaired glucose tolerance over the long term, but had no

62

effect on obesity in TSOD mice. Furthermore, we demonstrated that OPIACE mildly

63

reduced oxidative stress in TSOD mice when feeding duration was extended. Interestingly,

64

OPIACE also attenuated anxiety-like behavioral abnormality in aged TSOD mice. Our

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findings suggest that the oleuropein-rich diet has beneficial effects on the progression of

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type 2 diabetes and behavioral disorder that is likely related to diabetes.

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■ Materials and Methods

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Chemicals. 13-Hydroxy-9Z,11E-octadecadienoic

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acid acid

[13-(Z,E)-HODE],

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9-hydroxy-10E,12Z-octadecadienoic

[9-(Z,E)-HODE],

and

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13S-Hydroxy-10E,12Z-octadecadienoic-9,10,12,13-d4 acid (13-HODE-d4) were obtained

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from Cayman Chemical Company (MI, USA). 9-Hydroxy-10E,12E-octadecadienoic acid

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(9-(E,E)-HODE) and 13-hydroxy-9E,11E-octadecadienoic acid (13-(E,E)-HODE) were

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purchased from Larodan Fine Chemicals AB (Malmo, Sweden). OPIACE was provided by

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Eisai Food & Chemical Co., Ltd. (Tokyo, Japan). The nutrient composition in OPIACE

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was measured by high performance liquid chromatography (HPLC). OPIACE was

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dissolved in methanol/water (60:40, v/v) and subjected to HPLC analysis. Other materials

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were of the highest grade commercially available.

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Animals.

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Male TSOD mice and male TSNO mice (control) were obtained from the Institute

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for Animal Reproduction (Ibaraki, Japan). The animals were housed individually and had

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free access to food (CE-2; Clea Japan Inc., Tokyo, Japan) and water. The animal room was

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maintained at 23 ± 2 °C and 50 ± 10% humidity under a 12 h light (8:00-20:00) and dark

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(20:00-8:00) cycle. The animals were acclimated to the laboratory environment for at least

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one week before the experiment. TSOD mice were divided into two weight-matched

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groups (n=12 each); the control diet group and OPIACE-containing diet group. The

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OPIACE-containing diet was prepared by mixing the control diet (CE-2) with 0.2% (w/w)

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OPIACE, and began to feed TSOD mice from 4 weeks of age. The experimental protocols

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were approved by the Institutional Animal Care and Use Committee of the National 5



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Institute of Advanced Industrial Science and Technology.

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Measurements of body weight, food intake, and biochemical parameters.

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Body weight, food intake, and non-fasting blood glucose levels were measured

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once a week. Non-fasting blood glucose level was measured with the same method as that

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described previously4. For the measurement of plasma triglyceride levels, mice at 5, 8, 11,

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and 13 weeks of age were sacrificed after fasting for 16 h. Blood was collected from the

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vena cava and gently mixed with heparin. The obtained plasma samples were stored at -30

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°C until use. Plasma triglyceride levels were measured with a LabAssay Triglyceride kit

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(Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s

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instructions. The weight of epididymal white adipose tissue (WAT), a type of visceral fat, in

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mice was measured at 5, 8, 11, and 13 weeks of age.

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Oral glucose tolerance test (OGTT).

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After fasting for 16 h, glucose solution at the dose of 1.5 g/kg body weight was

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administered orally with a gavage needle to mice at 5, 8, 11, 13, 15, and 24 weeks of age.

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For the measurement of blood glucose level, mice were placed in a mouse restrainer and

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their tail tips were resected with surgical scissors. Glucose level in effused blood

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(approximately 1 µL) was measured with a glucose meter (Life Check; EIDIA Co., Ltd.,

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Tokyo, Japan). Measurement of blood glucose levels in tail was performed at 0

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(pre-administration), 15, 30, 60, and 120 min after glucose administration. The area under

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the curve (AUC) after OGTT was calculated using the trapezoidal method.

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Analysis of hydroxyoctadecadienoic acid by liquid chromatography-mass/mass

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spectrometry (LC-MS/MS).

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Hydroxyoctadecadienoic acids (HODEs) were measured with a previously

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described method23. Briefly, mouse plasma sample was mixed with the internal standard,

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13-HODE-d4, and then butylated hydroxytoluene and triphenylphosphine were added to

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reduce hydroperoxides in the sample. The reduced sample was saponified by adding KOH

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in methanol. The mixture was acidified with 10% (v/v) acetic acid in water and extracted

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with chloroform/ethyl acetate (4:1, v/v). The chloroform/ethyl acetate layer was

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concentrated and divided equally into two portions, and each portion was evaporated to

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dryness under nitrogen. One portion of the derivatized sample was reconstituted with

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methanol/water (70:30, v/v), and subjected to LC-MS/MS analysis of HODEs. The

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precursor, the product ions, and the collision energies were determined after the

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optimization of MS/MS conditions as follows: m/z = 295.0 and 194.6–195.6 at 21 eV for

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13-(E,E)-HODE; m/z = 295.0 and 170.5–171.5 at 24 eV for 9-(E,E)-HODE; m/z = 295.0

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and 182.6-183.6 at 22 eV for both 10-(Z,E)-HODE and 12-(Z,E)-HODE; and m/z = 299.0

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and 197.6–198.6 at 15 eV for 13-HODE-d4. Plasma total hydroxyoctadecadienoic acid

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(tHODE) levels were determined from the total amount of the products of the

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free-radical-mediated oxidation of the four isomers: 13-(Z,E)-HODE, 13-(E,E)-HODE,

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9-(Z,E)-HODE, and 9-(E,E)-HODE.

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Open field test.

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The open field test was performed according to a modified version of the test

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described previously24. The open field apparatus was constructed by a square box with a

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black wall (35×35×20 cm). The field was divided two areas: the central and peripheral

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zones. A central square (15×15 cm) was set in the middle of the open field, and another

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139

square was set in the peripheral area. A mouse was placed in one corner of the field and

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allowed to explore for 10 min. Locomotor activity (movement distance, line crossing, and

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movement rate) and anxiety-related parameters (freezing, entry into central area, and

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latency of entry into central area) were measured with the ANY-maze video tracking

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system (Brain Science Idea Co., Ltd., Osaka, Japan). The field was cleaned before the start

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of each session.

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Statistics.

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The results were expressed as means ± standard error. Outliers were omitted by

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the Smirnov-Grubbs’ outlier test. Statistical analysis was performed by using analysis of

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variance (ANOVA) followed by Tukey’s test for multiple comparisons with

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Ekuseru-Toukei 2012 (Social Survey Research Information Co., Ltd., Tokyo, Japan).

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Differences with a probability of 5% or less were considered significant.

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■ Results

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Effect of OPIACE on obesity-related parameters in TSOD mouse.

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We investigated the effect of OPIACE (the nutrient composition indicated in

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Table 1) on obesity, which is a major risk factor for the development of type 2 diabetes, in

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TSOD mice. As shown in Figures 2A and B, body weight and food intake of TSOD mice

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(black circle) were significantly higher than those of control TSNO mice (white circle)

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throughout the experiments. However, there were no significant differences in body weight

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and food intake between TSOD and OPIACE-fed TSOD (gray triangle) mice (Figures 2A

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and B). Whereas plasma triglyceride level and epididymal WAT weight, which are related

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to obesity, were significantly higher in TSOD mice (black bar) than in TSNO mice (white

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bar), these parameters were not significantly different between TSOD mice and

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OPIACE-fed TSOD mice (gray bar) (Figures 2C and D). The results indicate that

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OPIACE has no effect on obesity in TSOD mice.

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Effect of OPIACE on blood glucose level in TSOD mouse.

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Next, we investigated the effect of OPIACE on the diabetic phenotypes in TSOD

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mice. As shown in Figure 3, non-fasting blood glucose levels in TSOD mice (black circle)

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gradually increased with age, and a significant increase was noted from 6 weeks of age

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compared with age-matched TSNO mice (white circle). Whereas non-fasting blood glucose

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levels in OPIACE-fed TSOD mice (gray triangle) were comparable to those in TSOD mice

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until 9 weeks of age, glucose levels in the OPIACE-fed TSOD mice were significantly

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lower than those in the TSOD mice up to 24 weeks of age (Figure 3). The results indicate

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that OPIACE partially attenuates blood glucose elevation in TSOD mice over the long

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term.

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Effect of OPIACE on glucose tolerance in TSOD mouse.

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To evaluate the effect of OPIACE on glucose tolerance, we performed OGTT in

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TSOD mice at 5, 8, 11, 13, 15, and 24 weeks of age. At 5 weeks of age, there was no

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obvious abnormality in glucose tolerance of TSOD mice (black circle) compared with

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age-matched TSNO mice (white circle) (Figure 4). However, glucose levels after glucose

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loading in TSOD mice increased with age, and a significant increase was noted from 8

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weeks of age compared with age-matched TSNO mice (Figure 4), indicating that glucose

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tolerance in TSOD mice was impaired from around 8 weeks of age, consistent with our

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previous report25. Glucose tolerance in TSOD mice was markedly impaired after 11 weeks

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of age. On the other hand, the impaired glucose tolerance in TSOD mice at 11 and 24

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weeks of age was almost completely attenuated by OPIACE (gray triangle). Glucose levels

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after glucose loading in OPIACE-fed TSOD mice at 13 and 15 weeks of age tended to be

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lower than those of age-matched TSOD mice, although there were no significant

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differences between those glucose levels (Figure 4). Those findings indicate that the

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impaired glucose tolerance in TSOD mice is partially inhibited by OPIACE.

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Effect of OPIACE on oxidative stress in TSOD mouse.

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It is known that oxidative stress induces insulin resistance that in turn leads to the

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progression of type 2 diabetes5. To investigate the effect of OPIACE on oxidative stress in

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vivo, we examined plasma levels of tHODE, which has been used as a biomarker for the

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evaluation of oxidative stress23, in TSNO and TSOD mice. tHODE were determined from

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the total amount of the four isomers: 13-(Z,E)-HODE, 13-(E,E)-HODE, 9-(Z,E)-HODE,

199

and

9-(E,E)-HODE,

which

were

measured

with

LC-MS/MS

10


as

reported

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previously23(Figure 5A; also see Materials and Methods). As shown in Figure 5B, plasma

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tHODE levels in TSOD mice (black bar) were significantly higher than those in TSNO

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mice (white bar), consistent with our previous report4, and the levels were maintained

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during the measurements. Plasma tHODE levels in OPIACE-fed TSOD mice (gray bar)

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tended to decrease with feeding duration, and significantly decreased at 13 weeks of age

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compared with those in TSOD mice. The results indicate that OPIACE mildly attenuates

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oxidative stress in TSOD mice by long-term feeding.

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Effect of OPIACE on locomotor activity and anxiety-like behavior in TSOD mice.

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It has been reported that locomotor activity is reduced in type 2 diabetes model

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animals, including db/db mouse24, Akita mouse26, and high-fat fed rat27. The behavioral

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abnormality is more clearly observed in older animals with diabetes than younger

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animals24,27. In addition, repeated hyperglycemia was found to reduce locomotor activity

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in an open field test28, which is used to assess general locomotor activity and as an initial

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screen for anxiety-related behavior in rodents29. To investigate the long-term effects of

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OPIACE on the behavioral traits of TSOD mice, we performed an open field test in TSOD

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mice at 36 weeks of age. We found that the differences in body weight and food intake

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between TSNO and TSOD mice remained unchanged at 36 weeks of age (body weights of

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TSNO and TSOD mice were 37.8 ± 0.88 and 64.6 ± 1.24 g, respectively; food intake in

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TSNO and TSOD mice was 3.42 ± 0.13 and 4.64 ± 0.18 g/day, respectively), and that

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there were no significant differences in non-fasting blood glucose level and glucose

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tolerance between TSNO and TSOD mice (non-fasting blood glucose levels in TSNO and

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TSOD mice were 120 ± 0.85 and 136 ± 9.45 mg/dL, respectively; OGTT AUCs in TSNO

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and TSOD mice were 2.53 ± 0.09 and 3.08 ± 0.71 arbitrary units, respectively) (Table 2),

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224

as observed in other diabetic model ob/ob mice of a similar age30. Movement distance

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(Figure 6B), line crossing (Figure 6C), and movement rate (Figure 6D) were

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significantly decreased in TSOD mice (black bar) compared with TSNO mice (white bar),

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indicating that TSOD mice have low locomotor activity. OPIACE-fed TSOD mice (gray

228

bar) tended to attenuate the decreases in locomotor activity compared with TSOD mice

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(Figure 6B-D), although there were no significant differences in the results. TSOD mice

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showed increased freezing (Figure 6E), decreased frequency of entry into the central

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square (Figure 6F), and longer latency of entry into the central square (Figure 6G)

232

compared with TSNO mice, indicating that TSOD mice have anxiety-like phenotypes.

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Interestingly, the anxiety-like phenotypes in free-fed TSOD mice were significantly

234

attenuated by the feeding of OPIACE (Figure 6E and G). Thus, long-term feeding of

235

OPIACE has a preventive effect on the anxiety-like symptoms in type 2 diabetes model

236

mouse.

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■ Discussion

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Our results indicated that OPIACE attenuated chronic hyperglycemia (Figure 3)

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and impaired glucose tolerance (Figure 4) in TSOD mice. Those effects are likely to be due

240

to oleuropein because it is the major component in OPIACE (Table 1). It has been

241

demonstrated that an oleuropein-containing diet improved insulin sensitivity in middle-aged

242

overweight men31, and decreased homeostasis model assessment as an index of insulin

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resistance (HOMA-IR) in high-fat diet fed mice32. In addition, plasma insulin levels were

244

unchanged between TSOD mice and OPIACE-fed TSOD mice from 5 to 13 weeks of age

245

(unpublished data). These findings suggest that the improvement effects of OPIACE on

246

blood glucose level are due to not the acceleration of insulin secretion but the improvement

247

of insulin resistance.

248

As oleuropein has potent antioxidative activity in vitro17, we expected that the

249

preventive effects of an oleuropein-rich diet on blood glucose elevation in TSOD mice

250

would be partly due to the antioxidative capacity of oleuropein. However, OPIACE did not

251

clearly reduce plasma tHODE levels in TSOD mice even at 11 weeks of age, which is the

252

age when OPIACE exerted a preventive effect on the diabetic phenotypes (Figure 5B).

253

Furthermore, OPIACE had no effect on obesity in TSOD mice (Figure 2). It was reported

254

that potent antioxidants resveratrol33 and epigallocatechin-334 suppressed high-fat diet

255

induced obesity and hyperglycemia. In addition, in a previous DNA microarray analysis,

256

oleuropein was found to have beneficial effects on non-alcoholic fatty liver disease by

257

exerting multiple effects, including anti-lipogenesis, anti-inflammation, and anti-insulin

258

resistance35, and the multiple effects of oleuropein are likely to contribute to the

259

improvement of the diabetic phenotypes. Therefore, our results suggest that an

260

oleuropein-rich diet exerts not only a mild antioxidant effect but also other effects for the

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261

attenuation of the increased glucose levels in TSOD mice. A recent meta-analysis indicated that diabetes is associated with the increased

262 263

possibility of having anxiety disorders36. In fact, our results indicated that TSOD mice

264

exhibited anxiety-like behavior (Figure 6) consistent with other diabetic models, including

265

ob/ob mouse37, Akita mouse26, and high-fat fed mouse38. Some reports suggest that the

266

central nervous system disorder is a kind of diabetic complication9. It is easily presumed

267

that the comorbidity of diabetes and anxiety disorder may lead to a lower quality of life

268

than the morbidity of those diseases separately39. In addition, it was recently suggested that

269

the treatment of anxiety disorder may exacerbate the symptoms of diabetes, as selective

270

serotonin reuptake inhibitor, which is used for the treatment of mood and anxiety disorders,

271

promotes insulin resistance and insulin secretion in Min6 cells40. Thus, as shown in Figure

272

6, our results suggested that OPIACE may be a valuable supplement for reducing

273

diabetes-related anxiety, although the mechanism for attenuating the anxiety-like behavior

274

in TSOD mice is unknown. Blood glucose levels in TSOD mice and OPIACE-fed TSOD

275

mice at 36 weeks of age were not significantly increased compared with those in TSNO

276

mice, probably because food intake in TSOD mice tended to decrease with age (Table 2).

277

The improvement of blood glucose levels in TSOD mice over the long term might

278

contribute to the anti-anxiety effect of OPIACE.

279

In conclusion, we demonstrated that OPIACE attenuated chronic hyperglycemia

280

and impaired glucose tolerance in TSOD mice over the long term. Furthermore, OPIACE

281

attenuated the anxiety-like behavior in aged TSOD mice. Our results suggest that an

282

oleuropein-rich diet has the potential to inhibit the progression of type 2 diabetes and

283

diabetes-related behavioral disorder.

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■ Author information

285

Funding This work was supported by a Grant-in-Aid for Young Scientist (B) (Grant

286 287

Number 25860183) from the Japan Society for the Promotion of Science (JSPS), and by a

288

donation from Eisai Food and Chemical Co., Ltd.

289 290

Notes The authors declare no competing financial interest.

291 292 293

■ Acknowledgements

294

We thank Y. Fujita of AIST for technical assistance; Y. Senba of JAC Co., Ltd. for

295

supporting animal care; and Dr. W. Suzuki of Tsumura Co., Ltd., and Dr. K. Ohwada of

296

AIST for valuable suggestions and discussion.

297 298

■ Associated content

299

Supporting information This information is available free of charge via the Internet at http://pubs.acs.org.

300 301 302 303

■ Abbreviations used

304

OPIACE, olive leaf extract with more than 35% (w/w) oleuropein content; TSOD,

305

Tsumura Suzuki Obese Diabetes; TSNO, Tsumura Suzuki Non Obesity; WAT, white

306

adipose tissue; OGTT, oral glucose tolerance test; AUC, area under the curve; HPLC, high

307

performance liquid chromatography; HODE, hydroxyoctadecadienoic acid; LC-MS/MS,

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308

liquid chromatography-mass/mass spectrometry; ANOVA, analysis of variance.

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11. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N. Engl. J. Med. 1993, 329, 977-986. 12. Geijselaers, S. L.; Sep, S. J.; Stehouwer, C. D.; Biessels, G. J., Glucose regulation, cognition, and brain MRI in type 2 diabetes: a systematic review. Lancet Diabetes Endocrinol. 2015, 3, 75-89. 13. Wu, S. B.; Yue, G. G.; To, M. H.; Keller. A. C.; Lau, C. B.; Kennelly, E. J., Transport in Caco-2 cell monolayers of antidiabetic cucurbitane triterpenoids from Momordica charantia fruits. Planta Med. 2014, 80, 907-911. 14. Bhathena, S. J,; Velasquez, M. T., Beneficial role of dietary phytoestrogens in obesity and diabetes. Am. J. Clin. Nutr. 2002, 76, 1191-1201. 15. Servili, M.; Sordini, B.; Esposto, S.; Urbani, S.; Veneziani,G.; Maio, I. D.; Selvaggini R,; Taticchi, A., Biological Activities of Phenolic Compounds of Extra Virgin Olive Oil. Antioxidants 2014, 3, 1-23. 16 Omar, S. H., Oleuropein in olive and its pharmacological effects. Sci. Pharm. 2010, 78, 133-154. 17. Lee, O. H.; Lee, B. Y.; Lee, J.; Lee, H. B.; Son, J. Y.; Park, C. S.; Shetty, K.; Kim, Y. C., Assessment of phenolics-enriched extract and fractions of olive leaves and their antioxidant activities. Bioresour. Technol. 2009, 100, 6107-6113. 18. El, S. N.; Karakaya, S., Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr. Rev. 2009, 67, 632-638. 19. Park, S.; Choi, Y.; Um, S. J.; Yoon, S. K.; Park, T., Oleuropein attenuates hepatic steatosis induced by high-fat diet in mice. J. Hepatol. 2011, 54, 984-993. 20. Poudyal, H.; Campbell, F.; Brown, L., Olive leaf extract attenuates cardiac, hepatic, and metabolic changes in high carbohydrate-, high fat-fed rats. J. Nutr. 2010, 140, 946-953. 21. Grossi, C.; Rigacci, S.; Ambrosini, S.; Dami, T. E.; Luccarini, I.; Traini, C.; Failli, P.; Berti, A.; Casamenti, F.; Stefani, M., The polyphenol oleuropein aglycone protects TgCRND8 mice against Ass plaque pathology. PLoS One 2013, 8, e71702. 18


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22. Jemai, H.; El Feki, A.; Sayadi, S., Antidiabetic and antioxidant effects of hydroxytyrosol and oleuropein from olive leaves in alloxan-diabetic rats. J. Agric. Food Chem. 2009, 57, 8798-8804. 23. Yoshida, Y.; Kodai, S.; Takemura, S.; Minamiyama, Y.; Niki, E., Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples. Anal. Biochem. 2008, 379, 105-115. 24. Sharma, A. N.; Elased, K. M.; Garrett, T. L.; Lucot, J. B., Neurobehavioral deficits in db/db diabetic mice. Physiol. Behav. 2010, 101, 381-388. 25. Murotomi, K.; Umeno, A.; Yasunaga, M.; Shichiri, M.; Ishida, N.; Abe, H.; Yoshida, Y.; Nakajima, Y., Switching from singlet-oxygen-mediated oxidation to free-radical-mediated oxidation in the pathogenesis of type 2 diabetes in model mouse. Free Radic. Res. 2014, in press. 26. Maher, P.; Dargusch, R.; Ehren, J. L.; Okada, S.; Sharma, K.; Schubert, D., Fisetin lowers methylglyoxal dependent protein glycation and limits the complications of diabetes. PLoS One 2011, 6, e21226. 27. Erdos, B.; Kirichenko, N.; Whidden, M.; Basgut, B.; Woods, M.; Cudykier, I.; Tawil, R.; Scarpace, P. J.; Tumer, N., Effect of age on high-fat diet-induced hypertension. Am. J. Physiol. Heart Circ. Physiol. 2011, 301, H164-172. 28. Moghadami M.; Moghimi, A.; Ahangar, E.; Jalal, R.; Rassouli, M. B.; Shahri, N. M., Effects of infantile repeated hyperglycemia on behavioral alterations in adult male and female rats. Basic. Clin. Neurosci. 2012, 3, 60-67. 29 Prut, L.; Belzung, C., The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur. J. Pharmacol. 2003, 463, 3-33. 30 Genuth, S. M.; Przybylski, R. J.; Rosenberg, D. M., Insulin resistance in genetically obese, hyperglycemic mice. Endocrinology 1971, 88, 1230-1238. 31. de Bock, M.; Derraik, J. G.; Brennan, C. M.; Biggs, J. B.; Morgan, P. E.; Hodgkinson, S. C.; Hofman, P. L.; Cutfield, W. S., Olive (Olea europaea L.) leaf polyphenols improve insulin sensitivity in middle-aged overweight men: a randomized, placebo-controlled, crossover trial. PLoS One 2013, 8, e57622. 19



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32. Kim, S. W.; Hur, W.; Li, T. Z.; Lee, Y. K.; Choi, J. E.; Hong, S. W.; Lyoo, K. S.; You, C. R.; Jung, E. S.; Jung, C. K.; Park, T.; Um, S. J.; Yoon, S. K., Oleuropein prevents the progression of steatohepatitis to hepatic fibrosis induced by a high-fat diet in mice. Exp. Mol. Med. 2014, 46, e92. 33. Kim, S.; Jin, Y.; Choi, Y.; Park, T., Resveratrol exerts anti-obesity effects via mechanisms involving down-regulation of adipogenic and inflammatory processes in mice. Biochem. Pharmacol. 2011, 81, 1343-1351. 34. Bose, M.; Lambert, J. D.; Ju, J.; Reuhl, K. R.; Shapses, S. A.; Yang, C. S., The major green tea polyphenol, (-)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice. J. Nutr. 2008, 138, 1677-1683. 35. Kim, Y.; Choi, Y.; Park, T., Hepatoprotective effect of oleuropein in mice: mechanisms uncovered by gene expression profiling. Biotechnol. J. 2010, 5, 950-960. 36. Smith, K. J.; Beland, M.; Clyde, M.; Gariepy, G.; Page, V.; Badawi, G.; Rabasa-Lhoret, R.; Schmitz, N., Association of diabetes with anxiety: a systematic review and meta-analysis. J. Psychosom. Res. 2013, 74, 89-99. 37. Asakawa, A.; Inui, A.; Inui, T.; Katsuura, G.; Fujino, M. A.; Kasuga, M., Leptin treatment ameliorates anxiety in ob/ob obese mice. J. Diabetes Complications 2003, 17, 105-107. 38. Kaczmarczyk, M. M.; Machaj, A. S.; Chiu, G. S.; Lawson, M. A.; Gainey, S. J.; York, J. M.; Meling, D. D.; Martin, S. A.; Kwakwa, K. A.; Newman, A. F.; Woods, J. A.; Kelley, K. W.; Wang, Y.; Miller, M. J.; Freund, G. G., Methylphenidate prevents high-fat diet (HFD)-induced learning/memory impairment in juvenile mice. Psychoneuroendocrinology 2013, 38, 1553-1564. 39. Santos, M. A.; Ceretta, L. B.; Reus, G. Z.; Abelaira, H. M.; Jornada, L. K.; Schwalm, M. T.; Neotti, M. V.; Tomazzi, C. D.; Gulbis, K. G.; Ceretta, R. A.; Quevedo, J., Anxiety disorders are associated with quality of life impairment in patients with insulin-dependent type 2 diabetes: a case-control study. Rev. Bras. Psiquiatr. 2014, 36, 298-304.

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423 424 425

40. Isaac, R.; Boura-Halfon, S.; Gurevitch, D.; Shainskaya, A.; Levkovitz, Y.; Zick, Y., Selective serotonin reuptake inhibitors (SSRIs) inhibit insulin secretion and action in pancreatic beta cells. J. Biol. Chem. 2013, 288, 5682-5693.

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426

Figure captions

427

Figure 1. Chemical structure of oleuropein

428 429

Figure 2. Effects of OPIACE on obesity-related parameters

430

Time course of changes in body weight (A), food intake (B), plasma triglyceride

431

level (C), and epididymal WAT weight (D) in TSNO (white circle), TSOD (black circle),

432

and 0.2% (w/w) OPIACE-fed TSOD (gray triangle) mice. Results are expressed as means ±

433

standard error (n = 6-12). Statistical analyses were carried out using ANOVA (Tukey’s

434

post-hoc test). *Significant difference from age-matched TSNO mice (p < 0.05).

435 436

Figure 3. Effect of OPIACE on blood glucose level

437

Time course of changes in non-fasting blood glucose level in TSNO (white circle),

438

TSOD (black circle), and 0.2% (w/w) OPIACE-fed TSOD (gray triangle) mice. Results are

439

expressed as means ± standard error (n = 11-12). Statistical analyses were carried out using

440

ANOVA (Tukey’s post-hoc test). *Significant difference from age-matched TSNO mice (p

441

< 0.05). #Significant difference from age-matched TSOD mice (p < 0.05).

442 443

Figure 4. Effect of OPIACE on glucose tolerance

444

Time course of changes in glucose tolerance in TSNO (white circle), TSOD (black

445

circle), and 0.2% (w/w) OPIACE-fed TSOD (gray triangle) mice at 5, 8, 11, 13, 15, and 24

446

weeks of age. Results are expressed as means ± standard error (n = 11-12). Statistical

447

analyses were carried out using ANOVA (Tukey’s post-hoc test). *Significant difference

448

from age-matched TSNO mice (p < 0.05). # Significant difference from age-matched

449

TSOD mice (p < 0.05).

450 22


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451

Figure 5. Effects of OPIACE on plasma levels of lipid oxidation products

452

(A) Typical chromatograms obtained from LC-MS/MS of mouse plasma samples

453

measured in this study, (B) Time course of changes in the plasma levels of lipid oxidation

454

products in TSNO (white bar), TSOD (black bar) and 0.2% (w/w) OPIACE-fed TSOD

455

(gray bar) mice. Values in age-matched TSNO mice are set to 1. Results are expressed as

456

means ± standard error (n = 4-6). Statistical analyses were carried out using ANOVA

457

(Tukey’s post-hoc test). *Significant difference from age-matched TSNO mice (p < 0.05).

458

#Significant difference from age-matched TSOD mice (p < 0.05).

459 460

Figure 6. Effect of OPIACE on locomotor activity and anxiety-like behavior

461

(A) Representative trajectories of TSNO, TSOD, and 0.2% (w/w) OPIACE-fed

462

TSOD mice in the open field test. Movement distance (B), line crossing (C), movement rate

463

(D), freezing (E), entry into central square (F), and latency of entry into central square (G)

464

were measured for 10 min. Results are expressed as means ± standard error (n=6 each).

465

Statistical analyses were carried out using ANOVA (Tukey’s post-hoc test).

23



Table1. Nutrient composition of OPIACE Nutrient composition

(%)

Oleuropein

35

Phenolic compounds

7

Saccharides

54

Ignition residue

2

Moisture

2

Phenolic compounds consist of 0.08% caffeic acid, 0.06% vanillic acid, 0.04% vanillin, 0.26% rutin, 1.89% luteolin-7-glucoside, 1.04% apigenin-7-glucoside, and 3.8% oleuroside.

24


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Table2. Body weight, food intake, non-fasting blood glucose level, and OGTT AUC in TSNO, TSOD, and OPIACE-fed TSOD mice at 36 weeks of age TSNO

TSOD

Body weight (g)

37.8 ± 0.88

64.6 ± 1.24

Food intake (g/day)

3.42 ± 0.13

4.64 ± 0.18

5.19 ± 0.20

120 ± 0.85

136 ± 9.45

152 ± 32.5

2.53 ± 0.09

3.08 ± 0.71

2.69 ± 0.42

Non-fasting blood glucose (mg/dL) OGTT AUC (× ×104, arbitrary unit)

TSOD+OPIACE ∗ ∗

∗

65.2 ± 0.68

∗

Results are expressed as means ± standard error (n = 6 each). Statistical analyses were carried out using ANOVA (Tukey’s post-hoc test). ∗Significant difference from age-matched TSNO mice (p < 0.05).

25



Page 26 of 32

Figure 1

466

O

O

O

OH

OCH3

OH

O O

O OH

OH

OH

HO

26


Page 27 of 32


Figure 2

27



Page 28 of 32

Figure 3

Non-fasting blood glucose 400

TSNO

∗

TSOD

(mg/dL)

∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗

∗

∗

∗ ∗

200

∗

∗

TSOD+OPIACE

300

∗

∗ ∗

# # #

∗

∗#

# #

#

# #

#

100

0

4

6

8

10 12 14 16 18 20 22 24 Age (week)

28


Page 29 of 32


Figure 4

5 weeks of age

600

TSNO

500

500

TSOD

400

TSOD+OPIACE

300

∗

100

Blood glucose (mg/dL)

0 15 30

60

120

11 weeks of age

500

∗ ∗

400

#

500 400

∗ ∗

60

∗ ∗

∗ ∗

#

∗ ∗

200

#

∗

120

13 weeks of age

400 300

100

0 15 30

60

120

15 weeks of age

∗ ∗

∗

200

0

0 15 30

100

60

500

∗

400

∗

∗ #

300 200

#

100 0 15 30

60

120

120

24 weeks of age

600

∗ ∗

300

0

0 15 30

500

#

200

600

0

600

300

0

∗

200 100

600

100

∗

400 300

200

0

8 weeks of age

600

0

0 15 30

Time after glucose loading (min)

29


60

120


Figure 5

30


Page 30 of 32

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Figure 6

TSNO

(B)

TSOD

(C)

Distance

Line crossing

20

200

15

150

Frequency

(m)

p<

Oleuropein-Rich Diet Attenuates Hyperglycemia and Impaired

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