Flaxseed lignan secoisolariciresinol diglucoside improves insulin sensitivity through upregulation of GLUT4 expression in diet-induced obese mice

Journal of Functional Foods 18 (2015) 1–9

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Flaxseed lignan secoisolariciresinol diglucoside improves insulin sensitivity through upregulation of GLUT4 expression in diet-induced obese mice Yanwen Wang a,*, Bourlaye Fofana b,**, Moumita Roy a, Kaushik Ghose b, Xing-Hai Yao c, Marva-Sweeney Nixon d, Sandhya Nair a, Gregoire B.L. Nyomba c a Aquatic and Crop Resource Development, National Research Council of Canada, 550 University Avenue, Charlottetown, PE, Canada b Agriculture and Agri-Food Canada, 550 University Avenue, Charlottetown, PE, Canada c Section of Endocrinology & Metabolism, University of Manitoba, 715 McDermot Avenue, Winnipeg, MB, Canada d Department of Biology, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada

A R T I C L E

I N F O

A B S T R A C T

Article history:

The efficacy and mechanism of flaxseed lignan secoisolariciresinol diglucoside (SDG) on

Received 10 April 2015

glucose hemostasis in obese mice were examined. Male C57BL/6J mice fed on a high-fat diet

Received in revised form 19 June

(60 kcal% fat) for 15 weeks were divided into four groups and treated by oral gavage with

2015

0, 10, 100, or 1000 mg/kg/d of SDG dissolved in water for 6 weeks. Age-matched mice fed

Accepted 25 June 2015

with a low-fat diet (10% kcal fat) were used as the normal control. SDG lowered fasting blood

Available online

glucose, insulin, and free fatty acid levels and improved oral glucose tolerance, insulin response, and homeostatic model assessment-estimated insulin resistance index in mice fed

Keywords:

the high-fat diet. Flaxseed SDG also increased GLUT4 expression and tended to increase

Diet-induced obese mice

phosphorylated AKT to total AKT ratio in the muscle tissue. The data demonstrated that

Flaxseed SDG

flaxseed SDG improved glycaemic control, at least in part, by enhancing insulin signalling

Fasting blood glucose

and sensitivity in diet-induced obese mice.

Glucose tolerance

Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.

Insulin tolerance GLUT4

Chemical compound: Secoisolariciresinol diglucoside (PubChem CID: 9917980). * Corresponding author. Aquatic and Crop Resource Development, National Research Council of Canada, 550 University Avenue, Charlottetown, PE, Canada. Tel.: +1 902 566 7953; fax: +902 569 4289. E-mail address: [email protected] (Y. Wang). ** Corresponding author. Agriculture and Agri-Food Canada, 550 University Avenue, Charlottetown, PE, Canada. Tel.: +1 902 367 7602; fax: +902 566 7468. E-mail address: [email protected] (B. Fofana). Abbreviations: DIO, diet-induced obesity; FBG, fasting blood glucose; FFA, free fatty acids; HFC, high-fat diet control; HOMA-β, homeostatic model assessment of β-cell function; HOMA-IR, homeostatic model assessment of insulin resistance; LFC, low-fat diet control; L10, the high-fat diet supplemented with 10 mg/kg·d of flaxseed SDG; L100, the high-fat diet supplemented with 100 mg/kg·d of flaxseed SDG; L1000, the high-fat diet supplemented with 1000 mg/kg·d of flaxseed SDG; SDG, secoisolariciresinol diglucoside; TAG, triacylglycerols; TC, total cholesterol http://dx.doi.org/10.1016/j.jff.2015.06.053 1756-4646/Crown Copyright © 2015 Published by Elsevier Ltd. All rights reserved.

2

1.

Journal of Functional Foods 18 (2015) 1–9

Introduction

Plant lignans are polyphenolic compounds and ubiquitous within the plant kingdom (Tarpila, Wennberg, & Tarpila, 2005). Flax (Linum usitatissimum) seeds are among the richest sources of plant lignans (Mazur et al., 1996). Secoisolariciresinol diglucoside (SDG) is the major form of lignans found in flaxseed (Tourre & Xueming, 2010). Due to their oestrogenic activity, lignans belong to phytoestrogens and have been investigated for their influence on various hormonally sensitive end points, such as breast and prostate cancers (Adlercreutz, 2002; Adlercreutz, Heinonen, & Penalvo-Garcia, 2004; Truan, Chen, & Thompson, 2012). Other studies have shown that lignans and their metabolites, such as secoisolariciresinol, enterodiol and enterolactone, may protect against cardiovascular disease and metabolic syndrome by reducing lipid concentrations, lowering blood pressure, and decreasing oxidative stress and inflammation (Adolphe, Whiting, Juurlink, Thorpe, & Alcorn, 2010). Although the anti-diabetic effect of lignan SDG has been reported, limited information is available. A study assessed the effect of SDG on the development of diet-induced obesity in C57BL/6 mice and showed that a high-fat diet containing 0.5– 1.0% of SDG reduced visceral fat gain, and 1% SDG decreased serum insulin, leptin, total cholesterol and triacylglycerol concentrations compared with the high-fat diet without SDG (Fukumitsu et al., 2008), suggesting that SDG may improve insulin sensitivity. In contrast, a study in Zucker rats showed that SDG supplementation had no effect on the oral glucose tolerance (Prasad, 2001). In type 2 diabetic patients, lignan supplementation providing 360 mg SDG per day for 12 weeks reduced HbA1C concentration while having no effect on fasting glucose and insulin sensitivity (Pan et al., 2007). Another human study on metabolic syndrome showed that flax lignan supplementation providing 540 mg SDG per day for 6 months improved the metabolic composite score compared with placebo in male but not in female subjects (Cornish et al., 2009). Although flax lignan’s effects on diabetes are emerging, the efficacies are inconsistent. Moreover, few diabetes-related parameters were reported in the published studies (Cornish et al., 2009; Fukumitsu et al., 2008; Pan et al., 2007). A more comprehensive assessment using an increased number of parameters or biomarkers is therefore essential. Moreover, the mechanism of action of flaxseed lignans on glucose metabolism is not clear to date. The antioxidant and anti-inflammatory activities have been considered to contribute to the anti-diabetic benefits of lignans (Adolphe et al., 2010; Bhathena & Velasquez, 2002; Soltani, Washco, Morse, & Reisin, 2015). Whether lignans have any effect on insulin signalling has not been reported in most of the studies. Because the available literature did not clearly demonstrate the benefits of SDG, it is important to conduct additional studies looking into the efficacy and mechanism of action of this specific type of phytochemicals on glucose homeostasis. Accordingly, the present study was conducted to evaluate the glucose regulatory properties of flaxseed lignan SDG in mice with high-fat diet-induced obesity and insulin resistance. Here, we report that flaxseed lignan SDG significantly affects glucose homeostasis by reducing fasting blood glucose, insulin and free fatty acid

levels, suppressing perirenal fat deposition and improving insulin sensitivity and insulin signalling.

2.

Materials and methods

2.1.

Animal studies

Sixty male C57BL/6J mice fed a high-fat diet (60% energy from fat) starting at the age of 5 weeks and 11 male C57BL/6J mice fed a low-fat diet all the time were purchased from Jackson Laboratories (Jackson, FL, USA) at the age of 8 weeks. At arrival, they were housed individually in mouse cages with 12:12 h dark/light cycle and free access to water and the same high-fat (D12492) and low-fat (D12450B) diets (Research Diets Inc., New Brunswick, NJ, USA), respectively. After twelve weeks, they were weighed and tested for fasting blood glucose levels. Mice on the high-fat diet were then divided into four groups based on their blood glucose levels and body weights. One group was used as the high-fat diet control (HFC) and the other three were treated by oral gavage with 10 (L10), 100 (L100), and 1000 (L1000) mg/kg·d of flaxseed SDG lignans (95%, Changsha Huacheng Biotech Inc., Changsha, Hunan, China) mixed in water for 6 weeks, respectively. The mice fed the low-fat diet (LFC) were used as the normal control. Both LFC and HFC groups were gavaged with the control vehicle of water. During the treatment period, the low-fat diet group was switched from the commercial low-fat diet to a caseincornstarch-sucrose-based low-fat diet (AIN-93G). The highfat diet was based on the AIN-93G diet and modified to have 60 kcal% from fat (a mix of 96% lard and 4% sunflower oil). All experimental diets were prepared weekly in our lab and stored at 4 °C. Food consumption was monitored daily and mice were weighed weekly during the treatment period. At weeks 5 and 6 of treatment, oral glucose tolerance and insulin tolerance were tested. At the end, mice were fasted overnight (12 h) and blood was collected via cardiac puncture after anaesthesia with isoflurane inhalation. Serum was obtained by centrifugation and stored in cryogenic vials at −80 °C for later analysis of biomarkers. The left side medial thigh muscle was dissected, frozen immediately in liquid nitrogen, and stored in a −80 °C freezer for the measurement of protein expressions. The animal use and experimental procedures were approved by the Joint Animal Care and Research Ethics Committee of the National Research Council Canada in Charlottetown and the University of Prince Edward Island (animal use protocol #10-011). The study was conducted in accordance with the guidelines of the Canadian Council on Animal Care.

2.2.

Oral glucose tolerance test

Glucose tolerance test was performed during week 5 of treatment by orally administering glucose at a dose of 2 g/kg body weight after fasting (12 h). The blood glucose concentration was measured from tail vein blood at time 0, 30, 60, 90, and 120 min, respectively using an Accu-Chek glucometer (Roche Diagnostics, Toronto, ON, Canada).

Journal of Functional Foods 18 (2015) 1–9

2.3.

Insulin tolerance test

The insulin tolerance test was conducted during week 6 of treatment after semi-fasting (4 h) by giving 0.7 U/kg body weight of insulin through intraperitoneal injection. Blood glucose was measured from tail vein blood with the Accu-Chek glucometer at time 0, 30, 60, 90, and 120 min, respectively.

2.4.

Analysis of serum lipids

The fasting serum total cholesterol (TC) and triacylglycerols (TAG) were measured in triplicate by enzymatic methods on a Pointe-180 Analyzer (Pointe Scientific, Canton, MI, USA) (Wang et al., 2014). All reagents were provided by the analyser manufacturer.

2.5.

Measurement of serum free fatty acids

The fasting serum free fatty acids (FFA) were measured in duplicate using commercial kits (BioVision Research Products, Mountain View, CA, USA) following the kit instructions. Standards were prepared at a series of concentrations and run in parallel with the samples. The concentration of FFA was calculated in reference to the standard curve.

2.6.

Analysis of serum insulin

The fasting serum insulin was analysed using commercial ELISA kits (Crystal Chem, Downers Grove, IL, USA) following the kit instructions. The concentration was calculated in reference to a standard curve that was generated by running a series dilution of standard in parallel with the samples.

2.7.

centrifugation at 14,000 g at 4 °C for 20 min, the supernatants were collected and protein concentration was determined by the Bio-Rad protein assay reagent (Bio-Rad, Mississauga, ON, Canada).

2.10.

Western blot analysis

A total of 40 µg protein was separated on 12% polyacrylamide gel and transferred onto Immuno-blot PVDF membranes (Bio-Rad) using the Trans-Blot SD semi-dry Transfer System (BioRad). All the membranes were first blocked in 5% nonfat dry milk blotting grade blocker (Bio-Rad) in TBST and then incubated over night with rabbit monoclonal antibodies to PI3 kinase P85 with 1:900 dilution (v/v), AKT2 mouse monoclonal antibody with 1:1000 (v/v) dilution, and GLUT4 mouse monoclonal antibody with 1:1000 (v/v) dilution (Cell Signaling, Beverly, CA, USA), respectively. The blots were washed with 0.05% TBST for 5 times with 5 min each, and incubated for one hour with HRP conjugated goat anti-rabbit IgG secondary antibodies (1:50,000 dilution, v/v; Millipore, Billerica, MA, USA) for PI3 kinase and goat-anti mouse IgG secondary antibodies (1:3000 dilution, v/v; Bio-Rad) for GLUT4 and AKT2, respectively. The blots were then washed with 0.05% TBST for 6 times with 10 min each. Protein bands were developed with the Immun-Star™ WesternC™ Chemiluminescence Kit (Bio-Rad) and photographed with a ChemiDocXRS molecular imaging system (Bio-Rad). Mouse monoclonal antibody to β-actin (1:1000 dilution, v/v; Cell Signaling, Beverly, USA) and goat anti-mouse IgG secondary antibody (1:3000 dilution, v/v; Bio-Rad) were used to measure β-actin expression, which was used to evaluate the protein loading. Normalization was performed with the Image Lab software (Bio-Rad). Values are expressed in arbitrary densitometry unit of relative abundance in relation to the mean of the LFC.

Analysis of serum glucose 2.11.

The fasting serum glucose concentrations were measured using an enzymatic method at the beginning and end of the treatment. The reagents were purchased from Genzyme Diagnostics (Charlottetown, PE, Canada). The analyses were carried out in accordance with the manufacturer’s instructions.

2.8. Calculation of homeostatic model assessment of insulin resistance and β-cell function The homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as (glucose in mmol/L × insulin in mU/L)/22.5. The homeostatic model assessment of β-cell function (HOMA-β) was calculated as (20× insulin in mU/L)/(glucose in mmol/L − 3.5).

2.9.

3

Bio-plex protein assay

AKT and p-AKT were measured using the Bio-Plex protein assay kit following the manufacturer’s instructions (Bio-Rad). Briefly, 10 µg of protein from cell lysates of untreated Hela (positive control) for AKT, EGF-treated HEK293 for p-AKT Ser473 and Thr308, and tissue of experimental DIO mice were used in the assays. A blank consisting of only detection antibody dilution buffer was used as negative control. All procedures were performed by protecting the samples from light, at room temperature or 4 °C. Detection antibodies and streptavidinphycoerythrin (SA-PE) were used at 1:20 and 1:100 (v/v) dilutions, respectively. Fluorescence was read on a Bio-Plex system 200 (Bio-Rad) and sample fluorescence was expressed per unit of protein. p-AKT expression was expressed as a ratio of p-AKT fluorescence to that of AKT.

Muscle protein extraction 2.12.

Proteins were extracted from left-side medial thigh muscle tissues of 6 animals in each group. Frozen excised skeletal muscle was pulverized in liquid nitrogen and homogenized in ice-cold lysis buffer (pH 8) containing 40 mM Tris–HCl, 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]1-propane sulphonate, 1% dithiothreitol, 1 mM ethylenediaminetetraacetic acid, and protease inhibitor cocktail (Sigma-Aldrich, Oakville, ON, Canada) (Yuan et al., 2013). After

Statistical analysis

The data were analysed using the Student’s t-test for the difference between the LFC and HFC groups to demonstrate the difference between normal and obese mice. The treatment effects were assessed using one-way ANOVA. For body weight, food intake, fasting glucose, oral glucose tolerance and insulin tolerance, the effects were assessed using one-way ANOVA with repeated measures. When a significant treatment effect was

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Journal of Functional Foods 18 (2015) 1–9

detected, the least squares means test was used to determine the differences among the HFC, L10, L100, and L1000 groups. All statistical analyses were done using SAS software, version 9.2 (SAS Institute, Cary, NC, USA). The results are presented as means ± S.E.M., where a p value of less than 0.05 was considered significant.

3.3.

3.

Results

3.1.

Effect of flaxseed lignan SDG on mouse body weight

All animals behaved normally and none died due to the treatment. As shown in Table 1, the HFC group weighed significantly higher than the LFC group at the beginning of the experiment (p < 0.05), and this difference remained throughout the study period (p < 0.05). The dietary supplementation with flaxseed SDG at the dose of 10 mg/kg per day lowered the body weights at week 5 but had no effect in other weeks. The doses of 100 or 1000 mg/kg per day had no significant effect on the body weight in any week of the treatment period.

3.2.

have intrinsic ability to adjust food intake based on food energy density so as to maintain relatively constant energy intake. The supplementation with SDG at either dose did not affect the food intake in weeks 1, 2, 3 and 4 but significantly lowered food intake in week 5 (p < 0.05). The food intake was not measured in week 6.

Effect of flaxseed lignan SDG on mouse food intake

In contrast to the body weight changes, the food intake was significantly lower in the HFC group as compared to the LFC group (p < 0.05, Table 2). The effect was in accordance with energy density of the two diets (3850 kcal/kg of the low-fat diet vs. 5240 kcal/kg of the high-fat diet). Mice and other mammals

Effect of flaxseed SDG on fasting blood glucose

The fasting blood glucose was higher in the HFC group than the LFC group at the beginning of the experiment (p < 0.05) but did not differ after 6 weeks (Table 3). The fasting blood glucose levels in the three treatment groups were similar with the HFC group before the treatment. After 6 weeks, it was increased by 22% (p < 0.05) in the HFC group while no significant increases were observed in the three treatment groups.

3.4. Effect of flaxseed SDG on homeostatic model assessment estimated insulin resistance and β-cell function HOMA-IR and HOMA-β have been widely used to evaluate peripheral tissue insulin sensitivity and the function of pancreatic β-cells (Cacho, Sevillano, de Castro, Herrera, & Ramos, 2008; Wilson & Islam, 2012). In the current study, HOMA-IR index of the HFC group was increased over one-fold than that of the LFC. However, this effect was reversed by flaxseed SDG at any of the three doses (p < 0.05). Similar effects were observed on the HOMA-β at marginal significance level (p = 0.053).

Table 1 – Effect of flaxseed SDG on the body weight (g) of DIO mice. Treatment

LFC HFC L10 L100 L1000

Week 0

1

2

3

4

5

6

26.2 ± 0.6 29.3 ± 0.6† 28.9 ± 0.5 29.3 ± 0.5 29.3 ± 0.4

27.1 ± 0.8 30.5 ± 1.1† 28.1 ± 0.4 28.5 ± 0.5 28.7 ± 0.4

31.5 ± 0.2 34.5 ± 0.5† 32.2 ± 0.4 33.7 ± 0.3 33.3 ± 0.3

32.7 ± 0.3 36.3 ± 0.6† 33.1 ± 0.5 35.1 ± 0.4 34.5 ± 0.4

32.3 ± 0.3 37.2 ± 0.6† 34.4 ± 0.6 35.9 ± 0.4 35.3 ± 0.4

33.6 ± 0.3 38.7 ± 1.2†,a 34.8 ± 0.6a,b 37.1 ± 0.4a,b 36.3 ± 0.4b

32.6 ± 0.4 38.7 ± 0.7† 35.5 ± 0.5 37.8 ± 0.3 36.4 ± 0.5

The results are presented as means ± S.E.M. LFC, low-fat diet control; HFC, high-fat diet control; L10, L100, and L1000, the high-fat diet supplemented with 10, 100, and 1000 mg/kg·d of flaxseed SDG by oral gavage once a day, respectively. † Different from the LFC. a,b Values bearing different superscripts differ, p < 0.05.

Table 2 – Effect of flaxseed lignan SDG on the food intake (g) of DIO mice. Treatment

LFC HFC L10 L100 L1000

Week 1

2

3

4

5

5.0 ± 0.3 4.2 ± 0.6† 3.2 ± 0.4 3.9 ± 0.5 4.2 ± 0.3

4.7 ± 0.4 4.0 ± 0.3† 3.3 ± 0.1 3.6 ± 0.3 3.3 ± 0.2

3.6 ± 0.3 3.0 ± 0.2† 2.9 ± 0.1 3.4 ± 0.2 3.0 ± 0.1

3.8 ± 0.3 2.9 ± 0.2† 2.7 ± 0.2 2.7 ± 0.3 3.1 ± 0.4

3.6 ± 0.1 2.9 ± 0.3a,† 2.1 ± 0.1b 2.2 ± 0.2b 2.3 ± 0.1b

The results are presented as means ± S.E.M. LFC, low-fat diet control; HFC, high-fat diet control; L10, L100, and L1000, the high-fat diet supplemented with 10, 100, and 1000 mg/kg·d of flaxseed SDG by oral gavage feeding once a day, respectively. † Different from the LFC. a,b Values bearing different superscripts differ, p < 0.05.

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Journal of Functional Foods 18 (2015) 1–9

Table 3 – Effect of flaxseed lignans on fasting blood glucose and HOMA indexes in DIO mice. Treatment

FBG (mmol/L) Before treatment

FBG (mmol/L) After treatment

HOMA-IR

HOMA-B

LFC HFC L10 L100 L1000

7.0 ± 0.5 8.3 ± 0.4‡ 8.0 ± 0.4 8.2 ± 0.3 8.0 ± 0.4

9.1 ± 0.9† 10.1 ± 0.4† 8.9 ± 0.8 8.3 ± 0.7 8.3 ± 1.1

1.99 ± 0.39 4.66 ± 0.80‡,a 1.37 ± 0.22b 1.28 ± 0.17b 1.45 ± 0.27b

20.4 ± 3.31 32.59 ± 5.74 14.46 ± 2.84 16.04 ± 3.11 15.64 ± 3.41

The results are presented as means ± S.E.M. LFC, low-fat diet control; HFC, high-fat diet control; L10, L100, and L1000, the high-fat diet supplemented with 10, 100, and 1000 mg/kg·d of flaxseed SDG by oral gavage feeding once a day, respectively. † Different from the baseline (at the beginning of experiment). ‡ Different from the LFC. a,b Values bearing different superscripts differ, p < 0.05.

3.5. Effect of flaxseed SDG on serum insulin, lipids and free fatty acids The effect of flaxseed SDG on serum insulin and lipids is presented in Table 4. The serum insulin levels were significantly higher in the HFC group than the LFC group (p = 0.006), in agreement with the HOMA-IR, indicating that the high-fat diet induced insulin resistance in C57Bl/6 mice. After the treatment with flaxseed SDG for 6 weeks, the serum insulin levels were reversed (p < 0.0001) to the normal levels, with no differences being observed among the three SDG doses. A similar phenomenon was seen in the serum FFA levels. Mice in the HFC group showed higher serum concentrations of FFA than those in the LFC group (p = 0.0012). The SDG supplementation dose-dependently reduced the serum FFA. As a result, the L1000 group showed significantly lower serum FFA concentrations than the HFC group (p < 0.05). The total cholesterol levels were elevated in mice of the HFC group relative to the LFC group (p < 0.0001) but were not significantly altered by flaxseed SDG. Similarly, the serum triacylglycerol levels were higher in the HFC group than the LFC group at a marginal significance level (p = 0.0536) but no effect was seen for the supplementation with either dose of SDG.

3.6. Flaxseed SDG improves oral glucose tolerance in dietinduced obese mice All mice were tested for their glucose tolerance by orally administering glucose solution at a dose of 2 g/kg body weight

during week 5 of the treatment. As shown in Fig. 1A, there was a big increase of blood glucose in all groups at 30 min after the oral glucose administration and a substantial drop thereafter. As a result, after 60 min the blood glucose almost returned to the baselines except for the L100 group. SDG supplementation suppressed the elevation of blood glucose at 30 min. The L1000 group showed significantly lower glucose levels than the HFC group (p < 0.05), while L10 and L100 showed a trend but did not reach the significance level. The LFC group, however, showed a significant difference from the HFC group at 120 min after the oral glucose loading but not at any other time points. The area under the curve was not significantly different between the LFC group (3761 ± 73 mmol/L·min) and the HFC group (3860 ± 108 mmol/L·min).

3.7. Effect of flaxseed SDG on insulin tolerance in dietinduced obese mice There were significant differences in the blood glucose levels between the HFC and LFC at 60, 90, and 120 min after the intraperitoneal injection of insulin (Fig. 1B). Thus, mice of the HFC group were significantly less sensitive to the injected insulin. The blood glucose levels in the HFC group dropped 30 min post insulin injection but quickly returned to the baseline thereafter. A better and longer response was observed in the LFC group. There was a clear trend of improvement by flaxseed SDG supplementation, with significantly lower glucose levels observed in the L10 group 60, 90, and 120 min after the insulin injection.

Table 4 – Effect of flaxseed lignans on serum insulin and lipids of DIO mice. Treatment

Insulin (ng/ml)

TC (mg/dl)

TAG (mg/dl)

FFA (mmol/ml)

LFC HFC L10 L100 L1000

0.19 ± 0.2 0.42 ± 0.7†,a 0.14 ± 0.2b 0.14 ± 0.2b 0.14 ± 0.2b

122.6 ± 6.9 185.8 ± 8.5† 166.6 ± 8.6 177.2 ± 5.4 181.2 ± 5.8

58.1 ± 4.5 71.1 ± 4.9† 74.5 ± 3.7 76.1 ± 5.1 67.0 ± 3.1

1.70 ± 0.10 2.43 ± 0.16†,a 2.10 ± 0.14a,b 2.03 ± 0.16a,b 1.64 ± 0.19b

The results are presented as means ± S.E.M. LFC, low-fat diet control; HFC, high-fat diet control; L10, L100, and L1000, the high-fat diet supplemented with 10, 100, and 1000 mg/kg·d of flaxseed SDG by oral gavage feeding once a day, respectively. a,b Values bearing different superscripts differ. † Different from the LFC, p < 0.05.

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Journal of Functional Foods 18 (2015) 1–9

Fig. 1 – Effect of flaxseed SDG on oral glucose tolerance (A) and insulin tolerance (B) in high-fat diet-induced obese mice. (A) After 4 h of fasting, mice were orally gavaged with 2 g/kg BW of glucose in water and blood glucose was then measured with an ACCU-Chek glucometer at indicated time points from tail vein blood. #The L100 or L1000 had significantly lower blood glucose than the HFC; &the LFC was lower than the HFC, p < 0.05. (B) After 4 h of fasting, mice were injected intraperitoneally with 0.7 U/kg BW of insulin. Blood glucose level was measured at indicated time points from tail vein blood with an ACCU-Chek glucometer. #Different from the HFC group (p < 0.05). LFC, low-fat diet control; HFC, high-fat diet control; L10, L100, and L1000, the high-fat diet supplemented with 10, 100, and 1000 mg/kg·d of flaxseed SDG by oral gavage feeding once a day, respectively. All results are presented as means ± S.E.M.

3.8. Effect of flaxseed SDG on the protein expression of insulin signalling pathway The western blotting analysis showed that there were no differences between the HFC and LFC for the protein expression of PI3K, AKT2 and GLUT4 (Fig. 2A). The treatment of SDG had no effect on PI3k and AKT2 but increased GLUT4 expression compared to the HFC group (p < 0.05). Further analysis using the Bio-plex assay demonstrated that flaxseed SDG tended to increase the phosphorylation of AKT at serine 473 (p = 0.06), while having no significant effect on the AKT phosphorylation at threonine 308 (Fig. 2B).

Fig. 2 – Effect of flaxseed SDG on the protein expressions of the insulin signalling pathway. The expression of PI3K, AKT, and GLU4 was analysed using the Western blotting and the AKT phosphorylation by Bio-plex. The results are presented as means ± S.E.M. #Different from the HFC group, p < 0.05. LFC, low-fat diet control; HFC, high-fat diet control; L10, L100, and L1000, the high-fat diet supplemented with 10, 100, and 1000 mg/kg·d of flaxseed SDG by oral gavage feeding once a day, respectively.

3.9.

Effect of flaxseed SDG on visceral fat mass

As shown in Table 5, the HFC group had higher fat contents in both the epididymal and perirenal regions than the LFC group (p < 0.05). After 6 weeks of treatment, L10 or L1000 decreased the perirenal fat mass (p < 0.05) relative to the HFC group while no difference was seen between the L100 and the HFC. The differences disappeared after being calculated as the per cent of the body weight although the same trend was present.

4.

Discussion

Phytoestrogens have gained increasing attention in recent years because of accumulating evidence implying their protective roles against numerous metabolic disorders (Ayella et al., 2010; Bhathena & Velasquez, 2002; Pan et al., 2007; Truan et al., 2012). The health benefits of flaxseed or flaxseed lignans on glucose metabolism are less studied and there is lack of consistency among studies although the benefits have been reported (Adolphe et al., 2010). In patients with metabolic syndrome,

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Journal of Functional Foods 18 (2015) 1–9

Table 5 – Effect of flaxseed lignans on visceral fat mass of DIO mice. Treatment

Epididymal fat (g)

Perirenal fat (g)

Epididymal fat (% of BW)

Perirenal fat (% of BW)

LFC HFC L10 L100 L100

1.05 ± 0.08 2.41 ± 0.18† 2.12 ± 0.12 2.40 ± 0.11 2.08 ± 0.16

0.37 ± 0.04 1.01 ± 0.07† 0.84 ± 0.07‡ 0.93 ± 0.05 0.84 ± 0.05‡

3.20 ± 0.22 6.15 ± 0.36† 5.97 ± 0.29 6.32 ± 0.26 5.78 ± 0.47

1.14 ± 0.12 2.57 ± 0.13† 2.33 ± 0.15 2.46 ± 0.11 2.36 ± 0.17

The results are presented as means ± S.E.M. BW, body weight; LFC, low-fat diet control; HFC, high-fat diet control; L10, L100, and L1000, the high-fat diet supplemented with 10, 100, and 1000 mg/kg·d of flaxseed SDG by oral gavage feeding once a day, respectively. † Different from the LFC, p < 0.0001. ‡ Different from the HFC, p < 0.05.

supplementation of 543 mg SDG per day for 6 months decreased the metabolic syndrome composite score in males but not in females (Cornish et al., 2009). A recent study indicated that lignan metabolites, especially enterolactone, are associated with a lower risk of type 2 diabetes in U.S. women (Sun et al., 2014). Animal studies showed that SDG significantly prevented or delayed the onset of diabetes and improved glycaemic control in rats with either type 1 (Prasad, 2000; Prasad, Mantha, Muir, & Westcott, 2000) or type 2 diabetes (Prasad, 2001). In dietinduced obesity in C57BL/6 mice, a high-fat diet containing 1.0% SDG decreased serum insulin and leptin concentrations (Fukumitsu et al., 2008). In female Zucker rats, only two out of ten rats in the group receiving SDG developed glucosuria at the age of 72 d whereas all ten rats in the untreated group had glucosuria by this age (Prasad, 2001). SDG supplementation also reduced the incidence of diabetes in streptozotocin-induced diabetic rats, a type 1 diabetic condition (Prasad et al., 2000). The present study demonstrated that dietary supplementation of flaxseed SDG lignan suppressed the elevation of fasting blood glucose levels in mice with high-fat diet-induced obesity and insulin resistance. In agreement with the changes of fasting blood glucose levels, the high-fat diet-induced elevation of blood insulin concentrations was reversed by flaxseed SDG supplementation. The results suggest that flaxseed SDG lowered fasting blood glucose by improving insulin sensitivity. Additional supports were provided by the effects of SDG on the oral glucose tolerance, insulin tolerance, and calculated HOMA-IR index. Our further study on protein expression indicates that SDG likely improved insulin sensitivity by increasing AKT phosphorylation and GLUT4 protein expression. It is well known that glucose metabolism is regulated by insulin release from the pancreas and its action on the insulin signalling pathway in the peripheral tissues (Altaf, Barnett, & Tahrani, 2014). When blood glucose levels are elevated such as after a meal, pancreatic β-cells sense this change and release insulin immediately into the blood stream to upregulate the insulin signalling. However, when insulin resistance develops, more insulin is required to compensate the insulin resistance and a higher level of blood insulin occurs. The elevated fasting serum insulin levels in the high-fat dietinduced obese mouse clearly demonstrated this phenomenon. The reduction of insulin by flaxseed SDG indicates the improvement of insulin sensitivity. In addition to the commonly used glucose tolerance test, insulin tolerance test is also used

to evaluate insulin sensitivity in diabetes (Okita et al., 2014). In the present study, both tests were performed and the results consistently demonstrated the benefits of flaxseed SDG on blood glucose control and response to insulin. In comparison with diet-induced obese mice, flaxseed SDG improved glucose tolerance, especially at 30 min post oral glucose administration in mice treated with either 100 or 1000 mg/kg body weight per day. The SDG supplementation also improved the response to insulin injection in the insulin tolerance test, with the significant effect being observed in mice treated with 10 mg/kg·d while a consistent trend was shown in mice treated with the other two doses. A study in Zucker rats showed that SDG supplementation had no effect on the oral glucose tolerance (Prasad, 2001). The discrepancy might be due to the characteristic differences of the animal models. Zucker rats have a genetic defect and thus develop diabetes spontaneously while the high-fat diet-induced insulin resistance is due to obesity, an animal model that is relevant to the developmental aetiology of human insulin resistance and diabetes. Further evidence on the benefit of flaxseed SDG on insulin sensitivity was provided by the significant improvement of HOMA-IR index. In line with the present study, a clinical study in men and post-menopausal women, a dose of 40 g ground flaxseed-based product or matching wheat bran product for 10 weeks reduced HOMA-IR index (Bloedon et al., 2008). Similarly, a recent study in obese glucose intolerant people who consumed 40 g of ground flaxseed for 12 weeks showed significant improvement of the HOMA-IR index (Rhee & Brunt, 2011). In the current study, we have also calculated the HOMA-β index. The results showed consistently a trend of improving pancreatic β-cell function in mice treated with three different doses of SDG. The flaxseed SDG also reduced serum FFA although the serum TAG and TC levels were not significantly altered. A large body of evidence has shown that blood FFA is positively associated with insulin resistance (Soltani et al., 2015). Thus, reduction of serum FFA would benefit insulin function and glucose homeostasis, an additional supportive data to the observed improvement of insulin sensitivity by SDG. In agreement with the serum FFA reduction, SDG reduced the visceral fat mass, particularly in the perirenal region. This observation suggests that flaxseed SDG may improve insulin function by downregulating adiposity or increasing the clearance of free fatty

8

Journal of Functional Foods 18 (2015) 1–9

acids, possibly achieved by increasing its oxidation (Ashakumary et al., 1999). The molecular mechanism of action of lignans on glucose metabolism and homeostasis remains largely unclear. We showed that flaxseed SDG significantly increased the protein expression of GLUT4 in the muscle tissues. Although not significant, SDG showed a consistent trend of increasing the phosphorylation of AKT. These observations suggest that SDG may improve insulin sensitivity, at least in part, through the insulin signalling pathway. A previous study reported that the lignan-rich fractions of Fructus Schisandrae improved insulin sensitivity through the PPAR-γ pathway (Kwon, Kim da, Yang, & Park, 2011), which induces the expression of a number of genes involved in the insulin signalling cascade (Leonardini, Laviola, Perrini, Natalicchio, & Giorgino, 2009). Although not measured in the present study, flaxseed SDG is reported to have anti-oxidant and anti-inflammation properties (Bhathena & Velasquez, 2002). It is well studied that oxidative stress and inflammation are closely related to the development of diabetes and insulin resistance (Donath & Shoelson, 2011; Evans, Maddux, & Goldfine, 2005). The limitation of the present study is that mice in the lowfat diet group did not show significantly lower fasting blood glucose levels and showed similar oral glucose tolerance with the high-fat diet group. Since mice eat the majority of their food at night and have a high metabolic rate, overnight fasting prior to the oral glucose tolerance test might constitute a metabolic stress with impact on glucose (McGuinness, Ayala, Laughlin, & Wasserman, 2009). The high level of sucrose in the diet might be another contributory factor to the increase of fasting blood glucose over the treatment period and the impaired oral glucose tolerance in mice of the low-fat diet group. Importantly, all other parameters measured in the present study showed consistent improvements in mice supplemented with flaxseed SDG compared to mice fed the high-fat diet without SDG.

5.

Conclusion

The present study demonstrated that flaxseed SDG lignan had significant beneficial effects on glucose tolerance, insulin sensitivity, and possibly on the beta cell function as well. Protein expression studies indicated that improvement of glucose metabolism by flaxseed SDG might be, at least in part, due to the modulation of insulin signalling. The data collectively and consistently showed the benefits of flaxseed SDG on the management of circulating glucose in diet-induced obese mice, a model that mimics the conditions of human subjects with obesity and insulin resistance. Flaxseed lignan SDG appears to be a promising naturally-occurring therapeutic agent for insulin resistance and type-2 diabetes.

Conflict of interest The authors declare no conflict of interest.

Acknowledgements This research was part of the Total Utilization Flax Genomics (TUFGEN) project funded by Genome Canada/Genome Prairie (6003321) with financial contribution of Flax Council of Canada to BF. The authors want to thank Sarah Trash, and the animal technicians for their assistance and daily care of animals during the experiment. REFERENCES

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