Transgenic flax overexpressing polyphenols as a potential anti-inflammatory dietary agent

journal of functional foods 14 (2015) 299–307

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Transgenic flax overexpressing polyphenols as a potential anti-inflammatory dietary agent ☆ Magdalena Matusiewicz a,*, Iwona Kosieradzka a, Małgorzata Sobczak-Filipiak b, Magdalena Zuk c, Jan Szopa c a

Department of Animal Nutrition and Biotechnology, Faculty of Animal Sciences, Warsaw University of Life Sciences, Ciszewskiego 8, Warsaw 02-786, Poland b Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 159c, Warsaw 02-776, Poland c Department of Genetic Biochemistry, Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63/ 77, Wrocław 51-148 Poland

A R T I C L E

I N F O

A B S T R A C T

Article history:

The influence of chronic administration of seed cake of genetically modified flax W92/72,

Received 13 October 2014

which overexpressed polyphenols, on diet-evoked inflammation in the liver and other physi-

Received in revised form 3 February

ological disturbances was examined. High-fat-diet-induced rat obesity models were

2015

administered ad libitum the experimental diets (standard, high-fat with lard, and standard

Accepted 3 February 2015

and high-fat diets enriched with 30% seed cake of non-transgenic or transgenic flax) for

Available online

14 weeks. The beneficial effects of transgenic seed cake were related to carbohydrate metabolism, serum total antioxidant capacity and lipid peroxidation. The levels of the liver pro-

Keywords:

inflammatory cytokines tumor necrosis factor-α and interleukin-6 were decreased in the

Flax

transgenic and non-transgenic seed cake groups. Transgenic seed cake consumption el-

Flavonoids

evated the anti-inflammatory cytokines interleukin-4 and -10. The flaxseed cake groups had

SDG

improved liver ultra-structure. Diet supplementation with genetically modified flax W92/

GMO

72 seed cake may contribute to solving health problems resulting from high-energy diet

Inflammation

consumption. © 2015 Elsevier Ltd. All rights reserved.

Red-ox

1.

Introduction

Food fortification is the process of the addition of essential nutrients and health-promoting compounds (Espin, Garcia-Conesa, & Tomás-Barberán, 2007). Biofortification involves crop fortification at the source to synthesize and accumulate these

compounds (Gómez-Galera et al., 2010). Plants are able to be biofortified by modulating endogenous metabolic pathways, which increase the production of flavonoids (Wang, Chen, & Yu, 2011). An interesting subject of biofortification using genetic engineering methods is flax (Linum usitatissimum). It has resulted in the overexpression of polyphenolic antioxidants:

☆ This work is a part of Ph.D. thesis of Magdalena Matusiewicz. * Corresponding author. Department of Animal Nutrition and Biotechnology, Faculty of Animal Sciences, Warsaw University of Life Sciences, Ciszewskiego 8, Warsaw 02-786, Poland. Tel.: +48 692 628 961; fax: +48 22 593 66 69. E-mail address: [email protected] (M. Matusiewicz). http://dx.doi.org/10.1016/j.jff.2015.02.004 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

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journal of functional foods 14 (2015) 299–307

flavonoids, anthocyanins, phenolic acids and lignans in seeds which increased the protection against oxidation of the unsaturated fatty acids (UFA) (Lorenc-Kukuła et al., 2005; Z˙uk et al., 2011). Even after cold extraction of oil polyphenols remain associated with the flaxseed cake. Numerous studies have demonstrated that polyphenols are protective against pathologies caused by oxidative stress, such as metabolic disorders. Flavonoids and phenolic acids have demonstrated antioxidant and anti-inflammatory activities (Coppin et al., 2013; González et al., 2011; Martin et al., 2014; Moon, Yang, & Park, 2006; Soobrattee, Neergheen, Luximon-Ramma, Aruoma, & Bahorun, 2005; Wang et al., 2014). Flaxseeds are the richest dietary source of lignans; they contain secoisolariciresinol and trace quantities of matairesinol (Adlercreutz, 2007) which, during consumption, are converted to enterodiol and enterolactone. They have antioxidative and estrogen-like activities, which may lower chronic disease risks, including the risk for obesity (Adlercreutz, 2007; Prasad, 2000). A potentially useful target for polyphenol treatment is obesity, energy imbalance, a state of low-grade, chronic inflammation that promotes the development of insulin resistance and diabetes (Johnson, Milner, & Makowski, 2012). Inflammation is a common cause of liver disease (Berasain et al., 2009; Park et al., 2010). Innate immune system liver cells initiate and maintain liver inflammation through cytokine production (Liaskou, Wilson, & Oo, 2012). The objective of this study was to investigate the influence of chronic administration of seed cake of genetically modified (GM) flax with overexpressed polyphenols on the disturbances caused by the consumption of a high-fat diet, i.e., alterations in redox homeostasis, glucose and insulin concentrations, inflammatory state and the liver ultra-structure. To obtain the seed cake, oil was cold-pressed from flaxseeds (Z˙uk et al., 2011). The experimental dose of flaxseed cake was determined to not cause nutritional imbalances or metabolic disturbances in the experimental animals – rats (EFSA (Panel on Genetically Modified Organisms, GMO), 2011; EFSA (Scientific Committee), 2011).

2.

Materials and methods

2.1.

Plant material and transgenesis

Non-GM flax (Linum usitatissimum L. cv. Linola) seeds were obtained from the Flax and Hemp Collection of the Institute of Natural Fibers and Medicinal Plants (Poznan, Poland). The GM W92/72 line was received from the Department of Genetic Biochemistry of the University of Wroclaw (Wroclaw, Poland). The GM flax simultaneously overexpresses Petunia hybrida major enzymes of the flavonoid biosynthesis pathway: chalcone synthase (CHS), chalcone isomerase (CHI) and dihydroflavonol reductase (DFR). The expression of these enzymes improved antioxidant capacity of the seed extract which resulted in compositional changes for fatty acids and an increase in the production of quercetin and kaempferol derivatives, anthocyanins, phenolic acids (caffeic, ferulic, p-coumaric) and secoisolariciresinol diglucoside (SDG), the most important lignan component. The antioxidant capacity and bioactive

compound concentrations were also increased in the transgenic flaxseed cake extracts. The W92/72 flax production procedures and compositional details have previously been described (Lorenc-Kukuła et al., 2005; Z˙uk et al., 2011). The GM and non-GM plants were grown in a field in the vicinity of Wroclaw (trial No. 26, AM-13; Polish Environment Ministry No. 36/2011 decision, dated September 29th, 2011).

2.2. Nutritional experiment and animal material preparation An experimental model of obesity was used to determine the effect of a transgenic flaxseed cake diet on the development of diet-induced liver inflammation. Male Wistar–Crl:WI(Han) rats (The Center for Experimental Medicine, The Medical University of Bialystok, Poland) were fed a high-fat diet that was rich in pork lard (701 kcal GE/100 g, 456 kcal GE from crude fat/ 100 g) for 14 weeks, from the age of 5 weeks. The rats were maintained in individual growth cages (21 °C, 12 h/12 h, 40% humidity). For 14-week nutritional research the animals were divided into six groups (n = 6) with standardized body weights (bw) and were administered ad libitum: (1) standard diet (SD), (2) high-fat diet rich in lard (HFD), standard diet supplemented with 30% seed cake of either (3) the non-transgenic flax Linola (S Linola) or (4) the transgenic flax W92/72 (S W92), and high-fat diet supplemented with the same amount of seed cake of either (5) the non-transgenic flax (HF Linola) and (6) the transgenic flax W92/72 (HF W92) (Table 1). The diets were fabricated in the ‘Morawski’ Feed Production Plant (Kcynia, Poland). The standard diets met animal nutritional requirements; the high-fat diets substantially exceeded these requirements (NRC, 1996). The diet compositions and their nutritional values were determined according to the Association of Official Analytical Chemists (AOAC, 1996) procedures. The rats had free access to water. The body weight of animals and food intake were monitored weekly. After 12 hours of fasting, the rats were euthanized using an isoflurane (Aerrane, Baxter, Deerfield, IL, USA) overdose. Blood samples were collected from the heart in serum separating tubes or tubes containing lithium heparin for plasma separation. After blood clotting, the samples were centrifuged (10 min., 1006 ×g) and the serum was aliquoted and stored at −25 °C until further use. The erythrocytes were divided from the plasma using centrifugation (10 min., 1006 ×g, 4 °C) and were washed four times with 0.9% NaCl solution and stored at −25 °C. Liver tissue samples (right lobe) were stored at −80 °C, put into 4% formalin for histological analysis or into 2.5% glutaraldehyde in phosphate buffer pH 7.2 for transmission electron microscopic (TEM) observations. The experimental procedures were approved by the local ethics committee [Resolution No. 65/2010 of the III Local ethics committee on animal experiments in Warsaw, dated October 27th, 2010].

2.3. Serum glucose and insulin and a homeostatic model assessment of insulin resistance Serum glucose was determined using a photometric method on a Miura One analyzer (I.S.E. S.r.l., Rome, Italy). The serum insulin was determined using an enzyme-linked immunosorbent assay (ELISA, Wuhan EiAab Science Co., Ltd,

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Table 1 – Composition of experimental diets and their nutritional value. Component

Experimental diet

Flaxseed cake (%) Pork lard (%) Others

– – 30 – 44.7 – * ** *** Nutritional value (% dry matter) 19.2 17.3 22.2 3.4 48 11.5 8.9 2.3 5.9 62.6 28.2 53.4 470 701 516 32 456 109

SD

Crude protein Crude fat Crude fiber N-free extractives Gross energy (kcal/100 g) Gross energy from crude fat (kcal/100 g)

HFD

S Linola

S W92

HF Linola

HF W92

– ***

30 31.7 **

30 31.7 **

22.2 11.6 6.0 53.2 517 110

18.4 43 4.2 28.4 677 409

18.4 43 4.2 28.4 677 409

30

* Wheat, oats, fodder flour III, wheat bran, Hipro soybean meal, feeding yeast, fodder-chalk, fodder salt, Premix LRM. ** Hipro soybean meal, potato protein, monocalcium phosphate, fodder-chalk, fodder salt, feeding yeast, Premix LRM, fodder flour III. *** Fodder flour III, potato protein, Hipro soybean meal, feeding yeast, fodder-chalk, fodder salt, Premix LRM.

Wuhan, China). The homeostatic model assessment of insulin resistance (HOMA-IR) was determined (Matthews et al., 1985).

2.4.

Redox state

Serum total antioxidant activity was determined using the DPPH (2.2-diphenyl-1-picrylhydrazyl) method (Chrzczanowicz et al., 2008). The total antioxidant status (TAS) in liver was determined using a commercial kit (Randox, Crumlin, UK). Liver tissue samples were homogenized in cold 0.02 M phosphate-buffered saline (PBS) and centrifuged (30 min, 4 °C, 25 155 ×g) to collect the supernatant in which the protein concentrations were analyzed using a commercial kit based on Peterson’s modification of the Lowry method (Sigma, St. Louis, MO, USA). Superoxide dismutase (SOD) activity was evaluated in blood erythrocytes and liver tissue using a commercial kit (Randox, Crumlin, UK). The glutathione reductase (GR) activity was determined in blood serum using a commercial kit (Randox, Crumlin, UK). Lipid peroxides were evaluated in the serum and liver by measuring thiobarbituric acid reactive substances (TBARS), according to the Uchiyama and Mihara method (Uchiyama & Mihara, 1978).

2.5.

Inflammatory state of the liver

The inflammatory state biomarkers in the liver homogenates were determined using an ELISA (Wuhan EiAab Science Co., Ltd, Wuhan, China).

2.6.

Light microscopic evaluation of the liver

The fragments of liver were embedded in paraffin, sliced into 5 µm sections and stained using the hematoxylin-eosin (H&E) procedure. For visualization a light microscope (Leica DM750, Leica Microsystems Ltd., Heerbrugg, Switzerland) was used.

2.7.

with uranyl acetate and lead citrate and were evaluated using a JEOL JEM-1220 transmission electron microscope (TEM; JEOL Ltd., Tokyo, Japan).

Transmission electron microscopic liver evaluation

The pieces of the liver were post-fixed with osmium tetroxide (1%) and embedded in Epon. Ultrathin sections were stained

2.8.

Statistical analysis

Results are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) was performed with means compared using LSD test. A difference of P < 0.05 between the means was considered to be statistically significant. StatGraphics Centurion software (StatPoint Technologies, Inc., Warrenton, VA, USA) was applied.

3.

Results and discussion

3.1.

Body weight and food intake

The intake of the standard energy level diets was significantly higher than the high-energy diets (Fig. 1a). Animals that were fed the standard energy level diets containing flaxseed cake were characterized by significantly lower body weights compared with rats fed the high-fat diets (Fig. 1b). Studies that have investigated the effect of quercetin on body weight in obese rodents and humans have not produced definitive results. Rivera, Moron, Sanchez, Zarzuelo, and Galisteo (2008) demonstrated that in obese male Zucker rats, only a large dose of quercetin diminished body weight. Moreover, overweight-obese people who received quercetin had no changes in free-fat, fat mass, waist circumference and body weight (Egert, Boesch-Saadatmandi, Wolffram, Rimbach, & Muller, 2010). Furthermore, diet supplementation with ferulic acid suppressed body weight gain in C57BL/6J male mice fed a high-fat diet (Son, Rico, Nam, & Kang, 2010). The individual flavonoid and phenolic acid levels in the flaxseed cake (Lorenc-Kukuła et al., 2005; Z˙uk et al., 2011) used in the present study were smaller compared with the levels of the synthetic compounds used in the above-mentioned investigations.

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Fig. 1 – (a) Food intake and (b) body weight of rats fed different diets. Error bars indicate the standard error of the mean (SEM). Significant effect: values that do not share a common small letter (a, b) differ significantly at the level P < 0.05, and values that do not share a common capital letter (A, B) differ significantly at the level P < 0.01.

3.2. Serum glucose and insulin concentrations, and HOMA-IR The HFD-fed rats had increased level of glucose (Fig. 2). The addition of W92/72 seed cake to the diets had opposite effects (P < 0.01). A large serum insulin decrease and lower insulin resistance expressed as HOMA-IR were also observed in the S W92 and HF W92 groups compared with the other groups. These changes were statistically insignificant. Insulin resistance can be associated with obesity, type 2 diabetes and non-alcoholic fatty liver (Kim, Montagnani, Koh, & Quon, 2006). Quercetin reduced hyperinsulinemia and insulin resistance in obese rats but the plasma glucose was not affected (Rivera et al., 2008). SDG reduced diabetes development in a human diabetes model, Zucker diabetic fatty (ZDF)/Gmifa/fa rats, which may be associated with antioxidant activity of SDG metabolites (Prasad, 2000, 2001). In the study of Prasad (2001) cytokines [interleukin (IL)-6, tumor necrosis factor (TNF)α] are increased in type 2 diabetes and may stimulate macrophages and polymorphonuclear leukocytes to produce ROS. Increased glucose concentrations may also increase ROS generation through various mechanisms.

3.3.

The total antioxidant activity in serum, which represents the total antioxidant levels defending against free radicals and ROS, was expressed as DPPH radical scavenging activity which was higher (P < 0.05) in the rats that were administered diets with W92/72 seed cake addition compared with non-GM (Table 2). DPPH radical scavenging activity was higher in the HF W92 group than the SD (P < 0.05). The GR activity in the serum of rats that consumed the HF W92 diet was higher than HF Linola; these differences were not statistically significant. TBARS, the lipid peroxidation products, were measured to assess the oxidative stress degree and were lower in the serum of rats that were fed diets with flaxseed cake compared with HFD; these results were not statistically significant. The administration of the different diets did not influence the activity of the component of the antioxidant defense system, SOD, in erythrocytes and the liver. Higher values of liver TAS and GR activity were observed in rats fed the HF W92 diet compared with the HF Linola and HFD; these results were not statistically significant. However, the TBARS in the livers of HF W92-fed animals were elevated compared with HF Linola. These results were also not statistically significant and may relate to the total protein concentration. SDG has been demonstrated to diminish oxidative stress and lipid peroxidation and elevate antioxidant reserves in New Zealand White rabbits fed a cholesterol diet (Prasad, 1999). C57BL/KsJ-db/db mice, a model of type 2 diabetes mellitus, offered standard diet containing quercetin had elevated hepatic SOD activity and reduced TBARS (Jeong, Kang, Choi, Kim, & Kim, 2012). Mice fed a high-fat diet enriched with ferulic acid exhibited increased liver GR activity compared with the control and high-fat diet (Son et al., 2010). Plasma and erythrocyte TBARS were also reduced in these mice. Flaxseed supplementation reduced plasma TBARS in obese people intolerant to glucose (Rhee & Brunt, 2011).

3.4. Fig. 2 – Serum glucose and insulin concentrations and the homeostatic model assessment of insulin resistance (HOMA-IR) in animals fed the experimental diets. The error bars indicate the SEM. Significant effect: values that do not share a common letter (A, B) differ significantly (P < 0.01).

Redox state

Inflammatory state of the liver

The pro-inflammatory cytokines TNF-α and IL-6 in the livers of rats administered the diets containing flaxseed cake were markedly reduced compared with the HFD group; these changes were not statistically significant (Table 3). This trend was not

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Table 2 – The effect of experimental diet feeding on redox state indicatorsa. Concentration

Experimental diet

Serum DPPH radical scavenging (%) GR (U/L) TBARS (nmol/mL) Erythrocyte SOD (U/mg hemoglobin) Hepatic TAS (nmol/mg TP) SOD (U/mg TP) GR (U/mg TP) TBARS (nmol/mg TP) a

SD

HFD

S Linola

S W92

HF Linola

HF W92

SEM

P

5.14AB 55.31 0.94

8.37BC 133.38 1.44

4.14A 111.95 1.12

8.56BC 113.78 1.06

4.01A 91.69 1.05

10.01C 135.29 1.17

1.3510 23.7455 0.1734

0.0304 0.2371 0.4746

3.16

3.32

3.69

3.10

3.35

3.27

0.2312

0.5567

17.27 33.56 0.16 0.11

14.68 29.55 0.15 0.11

16.36 31.62 0.23 0.13

15.41 32.37 0.16 0.09

17.03 33.31 0.26 0.10

21.33 36.65 0.30 0.15

1.9158 3.6815 0.0536 0.0158

0.2265 0.8380 0.2925 0.1763

Significant effect: values within a factor that do not share a common superscript (A, B, C) differ significantly (P < 0.05).

observed for interferon-γ (IFN-γ) and C-reactive protein (CRP), an acute phase protein indicating chronic inflammation. These results may be associated with the tendency toward maintaining cytokine homeostasis. There were no differences in an anti-inflammatory adipocytokine adiponectin (ADP). The antiinflammatory cytokine IL-4 in the livers of rats fed S W92 was elevated compared with S Linola or the HFD (P < 0.01). IL-4 in the livers of rats that were administered a HF W92 did not differ from rats fed HF Linola and were improved compared with the HFD-fed (P < 0.01). There was a beneficial influence on anti-inflammatory IL-10 in animals fed a high-fat diet with flaxseed cake compared with HFD; this tendency remained statistically insignificant. Previous studies have demonstrated that the liver inflammatory pathways are primarily regulated by cytokines. TNF dependent signaling in the liver is important for the control of cellular homeostasis (Tacke, Luedde, & Trautwein, 2009). Serum and liver IL-6 are elevated in liver diseases (Tacke et al., 2009). Macrophages treated with quercetin had reduced IL-6 and TNF-α expression (Overman, Chuang, & McIntosh, 2011). Quercetin decreased macrophage mediated TNF-α expression in human adipocytes. These anti-inflammatory effects may be due to the interplay between oxidative stress and the inflammatory state. ROS are associated with the promotion of inflammatory processes by activation of transcription factors which induce the production of cytokines, such as TNF-γ (Rahman, 2002). Rivera et al. (2008) demonstrated that TNF-α production by visceral adipose tissue in obese rats was

increased compared with lean rats. The consumption of a high dose of quercetin diminished TNF-α production in obese rats. Plasma ADP was reduced in obese rats compared with lean rats. The intake of a high quercetin dose elevated ADP in obese rats. Stewart et al. demonstrated that a diet supplemented with quercetin lowered plasma IFN-γ in high-fat-diet-fed mice (Stewart et al., 2008). The administration of caffeic acid esters to Balb/c mice resulted to increased IL-4 in splenic lymphocytes that were stimulated in vitro by anti-CD3 antibodies (Park et al., 2004). Quercetin treatment in male Wistar rats that were administered ethanol increased serum IL-10 (Chen, 2010). The consumption of transgenic flavonoid-enriched tomatoes reduced CRP in humans and mice to a greater degree than was seen following the consumption of non-GM (Rein et al., 2006).

3.5.

Light microscopic evaluation of the liver

The livers from rats fed HFD (Fig. 3b) exhibited hepatocyte parenchymatous degeneration, microvesicular steatosis (numerous small fat vacuoles that do not displace a centrally placed nucleus) and hepatocyte disseminated necrosis. The livers of rats that were administered diets enriched with flaxseed cake were similar to rats fed a SD (Fig. 3 a, c, d, e, f). The number of cytoplasmic lipid vacuoles was lower, and only individual cells suffered from necrosis. Wistar rats administered a cholesterol diet that was SDGenriched along with secoisolariciresinol exhibited lower hepatic lipidosis (Felmlee et al., 2009). Quercetin reduced liver lipids

Table 3 – The influence of consuming different diets on the concentrations of pro- and anti-inflammatory state biomarkers in rat liversa. Concentration

TNF-α (pg/mg TP) IFN-γ (µg/mg TP) IL-6 (pg/mg TP) CRP (ng/mg TP) IL-4 (ng/mg TP) IL-10 (ng/mg TP) ADP (ng/mg TP) a

Experimental diet SD

HFD

S Linola

S W92

HF Linola

HF W92

6.31 5.28 7.95 0.20 19.14A 19.30 142.24

7.01 5.12 11.48 0.24 19.06A 16.07 137.05

4.20 4.97 5.46 0.18 20.24A 13.28 94.52

5.30 8.44 7.72 0.27 29.98B 23.10 142.78

4.93 7.14 7.79 0.21 24.54AB 22.45 138.75

5.28 8.09 7.31 0.38 31.31B 20.44 146.23

Significant effect: values within a factor that do not share a common superscript (A, B) differ significantly (P < 0.01).

SEM

P

0.6911 1.0314 1.3734 0.0538 2.6107 2.6276 21.1017

0.0918 0.0663 0.1026 0.1329 0.0038 0.0947 0.5279

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Fig. 3 – Morphological examination of the livers of rats that were administered different diets: (a) SD, (b) HFD, (c) S Linola, (d) S W92, (e) HF Linola, (f) HF W92. The liver sections were hematoxylin and eosin stained (H&E staining). Parenchymatous degeneration (PD), microvesicular steatosis (MS), necrosis (N). 20× magnification.

that were induced by a high-fat diet in male C57BL/6J mice, most likely due to the regulation of lipid metabolism gene expression and reduced biosynthesis of liver fatty acids and triacylglycerols (Jung, Cho, Ahn, Jeon, & Ha, 2013).

3.6.

TEM liver evaluation

Changes in organelle morphology were not observed (Fig. 4). In the hepatocytes of rats fed high fat diets (Fig. 4b, e, f), lipid

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305

Fig. 4 – Liver ultrastructural changes in rats fed the experimental diets: (a) SD, (b) HFD, (c) S Linola, (d) S W92, (e) HF Linola, (f) HF W92. Mitochondrion (M), rough endoplasmic reticulum (RER), sinusoidal lumen (SL), space of Disse (D), endothelial cell (EC), erythrocyte (ER), lipid droplet (L), collagen fibers (C), endothelial fenestrae (asterisk).

droplets of various sizes were present. In the liver tissue of rats receiving lower-fat-level feed that contained flaxseed cake, there was a decrease in lipid deposits to levels similar to the SD group (Fig. 4a, c, d). Vacuolization of certain cytoplasmic regions was visible in some individuals. The fibrosis rate, presence of collagen fibers in the space of Disse, which separates the hepatocyte by the cells of endothelium from sinusoidal lumen, was similar in all of the feeding groups. The sinusoidal area of rats fed the HFD was damaged, a thickening of the

sinusoidal endothelium and defenestration of endothelial cells were noted (Fig. 4b). The addition of flaxseed cake specifically to the standard energy level diets markedly improved the structure of the sinusoidal endothelium. Male C57BL/6J mice receiving a Western diet (high levels of fat and carbohydrates) demonstrated steatohepatitis and fibrosis, sinusoidal endothelium thickening, early capillarization, endothelial cell defenestration and the formation of subendothelial basal lamina (DeLeve, Wang, Kanel, Atkinson, &

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McCuskey, 2008). Thickened endothelium and defenestration may weaken the transfer of substrates between the sinusoid and hepatocytes, particularly that of lipoproteins (Jamieson et al., 2007). The liver sinusoidal endothelium is susceptible to oxidative stress. Its structure may be affected by the quantity of dietary load.

4.

Conclusions

The dietetic enrichment with GM flax W92/72 seed cake, which simultaneously overexpresses crucial enzymes in the flavonoid biosynthesis pathway (chalcone synthase, chalcone isomerase and dihydroflavonol reductase) and that is rich in flavonoids (quercetin, kaempferol), phenolic acids (caffeic, ferulic, p-coumaric), anthocyanins and secoisolariciresinol diglucoside may positively influence the development of an inflammatory state in an experimentally induced rat obesity model. These beneficial effects may be the result of improved carbohydrate metabolism, lower serum glucose and insulin and HOMA-IR. Furthermore, these effects are independent of diet energy level. Several indicators of redox homeostasis, serum total antioxidant capacity and lipid peroxidation state, expressed as TBARS, also indicated the beneficial influence of transgenesis. The pro-inflammatory cytokines TNF-α and IL-6 in the liver were diminished after the administration of flaxseed cake; these results also did not depend on diet energy level. W92/72 seed cake consumption increased anti-inflammatory cytokines IL-4 and IL-10. The ultrastructure of the liver was improved, lipid deposits were reduced, organelle appearance was maintained and the changes to the structure of the sinusoidal endothelium were ameliorated. Feeding rats a standard energy level diet that contained flaxseed cake effectively reduced body weight. This effect is most likely due to changes in the concentrations of biologically active substances after genetic transformation, complementary and synergistic polyphenol properties and the presence of compounds that were not a direct purpose of genetic modification, such as carbohydrate fractions and monounsaturated fatty acids. Diet enrichment with transgenic flax W92/72 seed cake may be a solution to the health issues that are a result of improper diet in humans and animals.

Conflicts of interest The authors declare no conflict of interest.

Acknowledgements This work was supported by the National Science Centre, Poland, Project No. 501-30-070400-A00512-99. REFERENCES

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