Flaxseed and quercetin improve anti-inflammatory cytokine level and insulin sensitivity in animal model of metabolic syndrome, the fructose-fed rats

1 Introduction

The metabolic syndrome (MetS) is a constellation of risk factors, including impaired fasting glucose, hypertension, central adiposity, predisposing to higher risks of oxidative stress, type 2 diabetes and atherosclerotic cardiovascular disease (CVD) (Park et al., 2007; Grattagliano et al., 2008;Chen et al., 2008 and Ishizaka et al., 2009). The etiopathology of the metabolic syndrome has not yet been fully elucidated. Recent studies have highlighted the involvement of a proinflammatory state that induces insulin resistance and leads to clinical and biochemical manifestations of the metabolic syndrome (Horiuchi and Mogi, 2011).

Obesity/insulin resistance is associated with metabolic syndrome, which plays a pivotal role in cardiovascular risk. The mechanisms that link obesity, insulin resistance, and endothelial dysfunction are numerous and complex (Steinberg et al., 1996). An increase in visceral fat, usually involved in obesity, leads to an imbalanced production of metabolic products, hormones, and adipocytokines including tumor necrosis factor-α (TNF-α), free fatty acids (FFAs) or adiponectin which causes decreased insulin sensitivity in skeletal muscle and liver, and impairs endothelial function through direct or indirect mechanisms.

Insulin itself acts as cytokine at sufficiently high concentrations and this may underlie vascular damage and dysfunction in human and animal studies (Absher et al., 1997, 1999). There are a number of recognized cytokines that are related to obesity, metabolic syndrome and cardiovascular disease, including adipocyte-related peptide adiponectin and inflammatory marker, interleukin-1b (IL-1b) (Dinarello, 1998; Huypens, 2007). Researchers observed that obesity is inversely correlated with adiponectin, a marker of anti-inflammation (Brooks et al., 2007). Similarly, IL-1b is a mediator of systemic pro-inflammatory pathways and may provide an index of the inflammatory processes that are known to accompany atherosclerosis.

The Mediterranean diet which includes a high intake of plant food content, such as vegetables, legumes, and fruits, has been directly associated with the prevention of obesity, type 2 diabetes, and other cardiovascular risk factors (Estruch et al., 2006). The protective effect of plant foods that contain flavonoids, polyphenolic compounds against chronic pathologies such as, obesity, diabetes and cardiovascular disease mortality is reported my many workers (Knekt et al., 2002; Mink et al., 2007).

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is one of the most widely used flavonols in human dietary sources (Hertog et al., 1993). The intervention trials with quercetin in human subjects are an effort in the development of dietary supplements with a higher dose, which might prove useful for the prevention or treatment of functional alterations clustered in the metabolic syndrome (Middleton et al., 2000; Duarte et al., 2002; Comalada et al., 2006; Rivera et al., 2008). Other human studies, however, failed to confirm these effects (Williamson and Manach, 2005).

Flaxseed is a complex food containing high amounts of poly unsaturated fatty acids (PUFA), mainly a-linolenic acid (ALA), an (n − 3) fatty acid, as well as soluble fiber, lignan precursors, and other substances that may have health benefits (Hall Iii et al., 2006). A number of studies have shown that flax oil supplementation can reduce serum triacylglycerols and cholesterol concentrations thus leading to reduced CVD risk (Cunnane et al., 1993 and Craig, 1999). Furthermore, n − 3 PUFA of flaxseed oil has anti-inflammatory properties that are mediated by the production of anti-inflammatory cytokines (Cohen et al., 2005). However, the effects of these foods on MtS remain unclear.

In this study, we determined the beneficial effects of quercetin, flaxseed and/or in combination as synergetic in an animal model of MtS, HF fed rats. In particular, we measured various parameters related to MetS, such as hyperlipidaemia, hyperglycemia, hyperinsulinemia and on formation of anti-inflammatory cytokines such as leptin and adiponectin.

2 Materials and methods

3 Biochemical assays

After an overnight fasting (food deprivation), rats were anesthetized and blood was withdrawn by heart puncture in tubes protected from light, then centrifuged at 3000g for 10 min at 4 °C. Plasma was immediately isolated, aliquoted, and stored at −80 °C until analyzed.

Serum glucose, total cholesterol, HDL-C, and TG levels were estimated calorimetrically using kits as cited in United Diagnostics Industry. LDL-C was calculated using the Friedwald equation [L = C−H−0.16T; where H is HDL-C, L is LDL-C, C is total cholesterol, T are TG, and k is 0.20 (mg/dl)]. Serum insulin, leptin and adiponectin were measured using enzyme linked immunosorbent assay (ELISA) kits, insulin [American Laboratory Products Company, Windham, NJ]; leptin and adiponectin [Ani Biotech Oy, Orgenium Laboratories Division, Vantaa, Finland). The reading was taken using ELISA microplate reader (VERSA Max, Molecular Devices Corporation, MN, USA).

4 Statistical analysis

All values are expressed as mean ± SE. Data were statistically analyzed using one way ANOVA for multiple group comparison, followed by Student’s unpaired t-test for group comparison. Significance was set at p ⩽ 0.05. Data were computed for statistical analysis by using Graph Pad Prism Software.

5 Results

After 4 weeks of feeding, a significant increase in serum glucose and insulin levels was observed in HF fed rats compared to the −ve control group. However, this increase was significantly decreased after administration of F (p ⩽ 0.001), Q (p ⩽ 0.05) and FQ (p ⩽ 0.001) in serum glucose (Fig. 1A). Serum insulin was significantly decreased after administration of F (p ⩽ 0.001) and FQ (p ⩽ 0.001) groups, whereas no significant difference could be observed in the Q group (Fig. 1B).

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Figure 1

Serum glucose and Insulin levels after administration of flaxseed, quercetin and in combination in fructose fed rats for 4-weeks. Values are expressed as mean ± SD. ^∗∗∗p ⩽ 0.001, ^∗∗p ⩽ 0.001, ^∗p ⩽ 0.05. (a) Compared to the (−ve) control group and (b) compared to the (+ve) control group.

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Serum lipid profiles were measured after 4 weeks in HF fed rats. Significantly higher concentrations of TG (P ⩽ 0.05), total cholesterol (P ⩽ 0.001) and LDL-C (P ⩽ 0.001) were observed in HF fed rats compared to the −ve control group, but these increases were significantly lowered after administration of F (p ⩽ 0.001), Q (p ⩽ 0.01) and FQ (p ⩽ 0.001) (Fig. 2A-C). A significant increase in serum HDL-C was observed in the FQ group (p ⩽ 0.001) compared to HF fed rats (Fig. 2D).

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Figure 2

Serum lipid profile level after administration of flaxseed, quercetin and in combination in fructose fed rats for 4-weeks. Values are expressed as mean ± SD. ^∗∗∗p ⩽ 0.001, ^∗∗p ⩽ 0.001, ^∗p ⩽ 0.05. (a) Compared to the (−ve) control group and (b) compared to the (+ve) control group.

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Serum leptin level, was decreased in F (p ⩽ 0.001), Q (p ⩽ 0.01) and FQ (p ⩽ 0.001) groups (Fig. 3A,) whereas serum adiponectin level, was increased in F (p ⩽ 0.001), Q (p ⩽ 0.01) and FQ (p ⩽ 0.001) groups (Fig. 3B).

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Figure 3

The leptin and adiponectin levels after administration of flaxseed, quercetin and in combination in fructose fed rats for 4-weeks. Values are expressed as mean ± SD. ^∗∗∗p ⩽ 0.001, ^∗∗p ⩽ 0.001, ^∗p ⩽ 0.05. (a) Compared to the (−ve) control group and (b) compared to the (+ve) control group.

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6 Discussion

Fructose rich diet was used for the induction of diabetes, which is characterized by insulin resistance and metabolic syndrome very much close to type 2 diabetes in humans. Several studies reported that fructose feeding for long term induces diabetes associated with insulin resistance and metabolic syndrome in experimental animals (Veerapur et al., 2010; Reungjui et al., 2007). In the present study, in rats receiving HF, plasma insulin, glucose, TG, total cholesterol and LDL-C were increased significantly, whereas HDL-C was significantly decreased compared to the −ve control group. These results are consistent with several earlier reports (Thresher et al., 2000; Busserolles et al., 2002, 2003) which support that consumption of HF diet leads to the development of insulin resistance which plays a pivotal role in the pathogenic mechanism of human type 2 diabetes and is the cause of all metabolic complications (Veerapur et al., 2010). High dietary fructose has been associated with enhanced oxidative damage in rats (Busserolles et al., 2002) and development of insulin resistance; beta-cell dysfunction, and impaired glucose tolerance (Paolisso and Giugliano, 1996; Bloch-Damti and Bashan, 2005).

In the present results, rats were fed HF diet, the cluster of metabolic syndrome was reversed by administration of F, Q and/or in combination (Fig. 1A and B). Some flavonoids, may affect fasting glucose, insulin, and lipid profile by insulin-enhancing activity in vitro and may regulate the expression of genes involved in glucose uptake and insulin signaling in rats fed with HF (Rivera et al., 2008). Epidemiological evidence indicated that flaxseed seemed to decrease fasting glucose, prevent the increase of glycolated hemoglobin (HbA1c) and delays the development of diabetes (Prasad, 2001). Castilla et al. (2006) found that consumption of grape juice, rich source of quercetin has an improving effect on HDL-C level which was paralleled by an increase in apo A1 concentrations. This indicates that flavonoids may affect hepatic apo A1 secretion in vivo (Hotamisligil et al., 1995).

Flaxseed contains both n − 3 fatty acids and lignans; flaxseed lignans alone had reduced serum lipids in hyperlipidaemic rats (Felmlee et al., 2009). Thus, the presence of lignans could have contributed to the lipid-lowering properties of flaxseed supplementation by inhibiting fatty acid and cholesterol synthesis in the liver and hence reducing the hepatic lipid levels (Fukumitsu et al., 2008). It may be attributable; in particular, that ALA-rich flax oil can act as a better substrate for mitochondrial and peroxisomal β-oxidation, thus stimulating increased oxidation of lipids in the liver (Ide et al., 2000). Thus flaxseed consumption could be predictive of benefits for people at risk of developing cardiovascular disease.

The results of the present study revealed a significant decrease in serum leptin and increase in adiponectin levels after administration of Q (p ⩽ 0.001), F (p ⩽ 0.05) and F + Q (p ⩽ 0.001) in HF fed rats (Fig. 3A and B). Quercetin has been shown to exert anti-inflammatory effects. In peripheral blood mononuclear cells quercetin dose-dependently inhibited the gene expression and production of the proinflammatory cytokine TNF-α (Nair et al., 2006). The mechanism of this effect was the modulation of the NF-kB signal transduction cascade. Therefore, a high dose of quercetin might have improved the inflammatory status, by decreasing the production of TNF- α in visceral adipose tissue, and increased plasma levels of adiponectin (Rivera et al., 2008).

The serum leptin level was lowered but serum adiponectin level was increased significantly in F and F + Q treatments (Fig. 3A and B). Flaxseed has recently gained popularity as a functional food. Consumption of flaxseed has been shown to lessen insulin resistance, hyperlipidemia, atherosclerosis and hypertension and decrease the incidence of cardiac arrhythmias. These effects of dietary flaxseed have been attributed, in part, to the rich ALA content of flaxseed (Ander et al., 2004; Dupasquier et al., 2007). However, the mechanism to explain the induction of these effects by ALA remains elusive. ALA in adipose tissue is strongly associated with increased leptin expression and subsequent reduction of atherosclerosis. Therefore, it is suggested that flaxseed may induce its anti-atherogenic effects in part via an ALA-mediated modulation of leptin expression (McCullough et al., 2011).

Adiponectin is the most highly expressed and secreted adipokine, with beneficial effects on metabolism, inflammation, and vascular function. It plays a role in insulin sensitivity, LDL oxidation, eNOS activation, inflammation suppression and fatty acid catabolism (Halleux et al., 2001; Haluzik et al., 2004; Iacobellis et al., 2005). Thus, hypoadiponectinemia is of interest as a biomarker of both cardiovascular disease and metabolic syndrome. A study conducted on mice, showed that replacing 15% of lipids with eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA] (specifically, the replacement lipid consisted of 6% EPA and 51% DHA) improved insulin sensitivity as well as raised plasma adiponectin level, independent of food intake or adiposity. It also showed that the adiponectin gene expression was up-regulated in mature adipocytes after this intervention (Albert et al., 2005).

7 Conclusion

These data are encouraging and suggest that anti-inflammatory and protective effect of flaxseed and quercetin consumption as functional foods could be an important mechanism contributing to the reduced risk for people with decreased insulin sensitivity and increased oxidative stress, such as those with the metabolic syndrome or type 2 diabetes.