Introduction

Along with changes in life style and diet structure, the prevalence of obesity is increasing globally. Many strategies have been attempted to control obesity and to prevent complications associated with it, including fatty liver, insulin resistance and cardiovascular diseases. Among strategies, supplementation of natural anti-oxidants is a promising one. For example, rutin, a potent natural anti-oxidant, has been demonstrated being capable of producing beneficial effects on several metabolic disorders (1–4). It has been reported that rutin suppresses high fat diet (HFD)-induced dyslipidemia in rats (1). Panchal et al. showed that rutin protects rats from the metabolic changes and cardiovascular remodeling caused by a high-fat and high-carbohydrate diet (2). Additionally, Hao et al. and Kamalakkannan et al. demonstrated that rutin improves the metabolic status of diabetic rats (3,4). Similarly, chronic inflammation has been indicated a critical role in development of metabolic disorders (5,6). For example, Henao-Mejia et al. reported that chronic inflammation is essential for progression of obesity and non-alcoholic fatty liver (5). A more recent study by Stienstra et al. identified a crucial function of inflammasome in pathogenesis of obesity and insulin resistance (6). Importantly, Hu et al. demonstrated that rutin ameliorates inflammasome activation, leading to suppressed inflammation in the kidney of rats (7). Similarly, Javed et al. reported the activity of rutin in preventing cognitive impairments in rats (8). Results from these studies prompt us to hypothesize that the beneficial effect of rutin in obesity is achieved through suppression of chronic inflammation.

Employing both in vitro and in vivo systems, we demonstrate in this study that rutin suppresses palmitic acid-triggered inflammation in macrophages, and twice weekly injections of rutin protect mice from development of HFD-induced obesity, fatty liver and insulin resistance. These beneficial effects are correlated with blockade of macrophage infiltration and chronic inflammation in adipose tissue. Our results offer new and critical information regarding the beneficial effects of rutin in the management of metabolic disorders.

Materials And Methods

Animals and Treatments

The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Georgia (Protocol Number, A2011 07-Y2-A3). Male C57BL/6 mice purchased from Charles River (Wilmington, MA) were housed under standard conditions with a 12 h light–dark cycle. Mice were divided into 3 groups (n = 5), and animals in group one were intraperitoneally (i.p.) injected twice weekly with rutin (50 mg/kg) solubilized in DMSO and fed a HFD (60% calories from fat, 20% from carbohydrates, and 20% from proteins). Mice in group two and three were injected (i.p.) with carrier solution fed a HFD or regular chow (20% calories from fat, 60% from carbohydrates, and 20% from proteins), respectively. Body weight and food-intake were measured weekly. Body composition analysis using EchoMRI-100 (Echo Medical Systems, Houston, TX) was conducted at the end of treatment.

Cell Culture

Murine macrophage cell line (RAW 264.7, TIB-71 at ATCC) was cultured at 37°C in a 5% CO2-humidified incubator and grown in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml). Cell viability was measured by MTT assay. For TNFα determination in supernatants, cells were pre-treated with various concentrations of rutin for 4 h and further incubated with 400 μM palmitic acids with or without rutin for 12 h. Supernatants were collected and TNFα levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit (#88-7324-88) purchased from eBioscience (San Diego, CA). In the gene expression analysis, cells were pre-treated with or without 200 μM rutin for 4 h and further incubated for 12 h with 400 μM palmitic acids with or without the presence of rutin (200 μM).

Measurement of Reactive Oxygen Species (ROS)

ROS was determined using the Total ROS Detection Kit (ENZ-51011) from Enzo Life Sciences (Farmingdale, NY). Cells were pre-treated with or without 200 μM rutin for 4 h and further incubated with 400 μM palmitic acids with or without 200 μM rutin for 24 h. Cells were washed with phosphate buffered saline (pH = 7.2) and incubated with the detection reagent from the kit for 30 min at 37°C. Cells were examined under a fluorescent microscope (ECLIPSE Ti, Nikon) and fluorescent intensity from at least 5 randomly selected fields was measured and normalized to cell numbers using the NIS-Elements imaging platform from Nikon Instruments Inc. (Melville, NY).

Intra-Peritoneal Glucose Tolerance Test (IPGTT) and Insulin Tolerance Test (ITT)

Mice were fasted for 6 h before IPGTT. Glucose solubilized in phosphate buffered saline was injected (i.p.) at 1.5 g/kg, and the time-point was set as 0 min. Blood glucose levels at 0, 30, 60 and 120 min were measured using glucose test strips and glucose meters. For ITT, mice were fasted for 4 h before the injection of insulin (Humulin, 0.75 U/kg) from Eli Lilly (Indianapolis, IN), and blood glucose levels were measured at 0, 30, 60 and 120 min after insulin injection.

Gene Expression Analysis

Total mRNA from the liver was isolated using the TRIZOL reagent from Invitrogen (Grand Island, NY). A commercial kit (#74804) from QIAGEN (Valencia, CA) was used for mRNA isolation from adipose tissue. Reverse transcription polymerase chain reaction (RT-PCR) was performed using a Superscript RT III enzyme kit (#11752-050) from Invitrogen. Quantitative real time PCR (qPCR) was conducted using SYBR Green as detection reagent and the data were analyzed using the ΔΔCt method (9). GAPDH mRNA level was used as an internal control, and all primer sequences used are listed in Table I. Primers were synthesized at Sigma (St. Louis, MO). Melting curve analysis of all real time PCR products were conducted and showed a single DNA duplex.

Table I PCR Primer Sequences
Full size table

Determination of Blood Concentrations of Insulin, Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT)

Blood samples were collected from heart cavities immediately after euthanizing mice. Serum was isolated by centrifugation at 4,000 rpm for 5 min. Insulin levels in the blood were measured using a commercial ELISA kit (10-1113-01, Mercodia Developing Diagnostics). AST (TR70121) and ALT (TR71121) concentrations were determined following manufacture’s instruction (Thermo-Scientific, Middletown, VA).

Determination of Triglyceride Level in the Livers

The measurement was performed according to a previously reported procedure (10). Briefly, the liver was freshly cut at ~200 mg per piece and homogenized using a solution consisting of chloroform and methanol (2:1). The homogenates were incubated at 4°C overnight and centrifuged at 12,000 rpm for 20 min. Supernatants were collected, dried and re-dissolved in 5% Triton-X100. The triglyceride levels were determined using a commercial kit (TR22203) from Thermo-Scientific (Middletown, VA).

H&E Staining

Tissues were freshly collected after euthanizing mice. Tissue samples were fixed, dehydrated and embedded into paraffin. Tissue sections were cut at 6 μm in thickness and dried at 37°C for 1 h. H&E staining was performed following the instruction of a commercial kit (#3500) from BBC Biochemical (Atlanta, GA). Slides were examined using an optical microscope and size measurements of adipocytes were taken using the NIS-Elements imaging platform purchased from Nikon Instruments Inc. (Melville, NY).

Oil-Red O Staining

Liver samples were freshly collected and immediately frozen in liquid nitrogen. Tissue sections were cut at 8 μm in thickness using a Cryostat. Sections were placed on slides and fixed using neutral buffered formalin for 30 min. The sections were washed with 60% isopropanol before being stained with freshly prepared Oil-red O working solution (#26079-05, Electron Microscopy Sciences) and counterstained with haematoxylin.

Statistics

Statistical analysis was conducted using one-way analysis of variance, and a P value below 0.05 (P < 0.05) is considered significantly different. The data were expressed as the mean ± SD.

Results

Rutin Suppressed the Palmitic Acid-Triggered Production of TNFα by Macrophages

We first determined the suppressive effect of rutin (Fig. 1a) on palmitic acid-triggered release of TNFα from macrophages. Palmitic acids induced TNFα release in a concentration-dependent manner. The TNFα level in culture medium was 900 pg/ml at palmitic acid concentration of 400 μM without affecting the viability of cells (Fig. 1b). Pre-treatment with rutin at 400 μM did not affect the viability of cells but suppressed the elevated production of TNFα by ~33% (Fig. 1c).

Fig. 1
figure 1

Suppression effect of rutin on palmitic acid-triggered release of TNFα by macrophages. (a) Molecular structure of rutin. (b) Palmitic acid concentration dependent TNFα release. RAW 264.7 macrophages cultured in DMEM were incubated with different concentrations of palmitic acid for 12 h and supernatants were collected for TNFα determinations using ELISA kit. (c) Rutin suppressed the palmitic acid-induced release of TNFα in macrophages. RAW 264.7 macrophages were pre-treated with various concentrations of rutin for 4 h and then incubated with 400 μM palmitic acids with or without rutin for 12 h. Supernatants were collected for determination of TNFα concentration. ** P < 0.01 vs control; ## P < 0.01 vs cells treated with palmitic acids without rutin. Data were from 3 independent experiments.

Full size image

Rutin Decreased Transcription of Genes for Inflammation and ER Stress in Macrophages

We next determined the mRNA levels of genes involved in inflammation and ER stress in macrophages. Palmitic acids increased the expression of Tnfα by ~2.3-fold and pre-treatment with rutin repressed the elevation by ~64.7% (Fig. 2a). Similarly, palmitic acids elevated the transcription of genes critical for inflammation including Ifnγ, Il1β, Il6 but not Mcp1, and pre-treatment with rutin greatly decreased these elevations (Fig. 2b). Moreover, palmitic acids increased the expression of Gpr94, a gene important for ER stress, by ~4.2-fold and pre-treatment with rutin suppressed the elevation by ~85.2% (Fig. 2c). Additionally, palmitic acids also slightly increased the expression of other genes involved in ER stress such as Xbp1, Ede1 and Herp, and the elevations were completely blocked by pre-treatment with rutin (Fig. 2d).

Fig. 2
figure 2

Suppression effect of rutin on palmitic acid-induced elevation of gene expression involved in inflammation and ER stress. RAW 264.7 macrophages were pre-treated with or without 200 μM rutin for 4 h and then incubated with 400 μM palmitic acid and/or 200 μM rutin for 12 h. (a) Relative mRNA levels of Tnfα. (b) Relative mRNA levels of genes involved in inflammation. (c) Relative mRNA levels of Gpr94. (d) Relative mRNA levels of genes involved in ER stress. ** P < 0.01 vs control; ## P < 0.01 vs cells treated with palmitic acid without rutin. Data were from 3 independent experiments.

Full size image

Rutin Repressed ROS Production in Macrophages

To explore the possible mechanism, we next examined the production of ROS in macrophages. Palmitic acids markedly increased the intracellular ROS in macrophages, and pre-treatment with rutin significantly suppressed this elevation (Fig. 3a). Treatment of cells with rutin alone did not have any effect. Quantitative analysis shows that palmitic acids increased the signals of ROS by ~5.2-fold, and pre-treatment with rutin reduced this elevation by ~68% (Fig. 3b).

Fig. 3
figure 3

Suppression effect of rutin on palmitic acid-caused production of ROS in macrophages. RAW 264.7 macrophages were pre-treated with or without 200 μM rutin for 4 h and further incubated with 400 μM palmitic acid and/or 200 μM rutin for 24 h. Cells were washed with PBS and incubated with the detecting reagent from the Total ROS Detection Kit for 30 min at 37°C. (a) Representative pictures of ROS detection. (b) Quantitative analysis of ROS production (* P < 0.05 vs control; ** P < 0.01 vs control; ## P < 0.01 vs cells treated with palmitic acids without rutin).

Full size image

Rutin Protected Mice from HFD-Induced Obesity

We evaluated the anti-obese activity of rutin in male C57BL/6 mice fed a HFD. Compared to mice fed a regular chow, Fig. 4a shows HFD-fed mice had a significantly high body weight at ~39.2 g. Twice weekly intra-peritoneal injections of rutin completely blocked HFD-induced weight gain. The difference between HFD-fed mice with or without rutin injection was ~9.8 g and can be recognized visually (Fig. 4b). To investigate the impact of the treatment on body composition, we measured lean and fat mass. Rutin greatly suppressed the increase of fat mass with no significant impact on lean mass (Fig. 4c). In addition, treatment with rutin did not show significant impact on food-intake in mice on HFD (Fig. 4d).

Fig. 4
figure 4

Effect of rutin on body weight and food-intake of animals fed a HFD. (a) Growth curve (n = 5, ** P < 0.01 vs mice fed with regular chow; ## P < 0.01 vs mice fed with HFD). (b) Representative pictures of mice (Bar length = 1 cm). (c) Body composition (n = 5, ** P < 0.01 vs mice fed with regular chow; ## P < 0.01 vs mice fed with HFD). (d) Average food intake.

Full size image

Rutin Prevented HFD-Induced Hypertrophy in Adipocytes

HFD feeding for 8 weeks increased the weight of the epididymal white adipose tissue (WAT) by ~4.1-fold, and rutin completely blocked the increase (Fig. 5a). Interestingly, neither HFD feeding nor rutin treatment showed impact on brown adipose tissue (BAT) (Fig. 5a). H&E staining of adipose tissues show that HFD-feeding caused apparent hypertrophy in adipocytes of WAT and also increased fat accumulation in BAT (Fig. 5b). The hypertrophy in WAT was associated with an increase in crown-like structures (arrows in Fig. 5b), a sign of macrophage activation. Rutin injection completely blocked the developments of hypertrophy and formation of crown-like structures in WAT (Fig. 5b). Quantitative analysis of white adipocytes shows the same conclusion (Fig. 5c).

Fig. 5
figure 5

Rutin-mediated blockade of hypertrophy and crown-like structures in the WAT of mice fed a HFD. (a) Weight of adipose tissues (n = 5, ** P < 0.01 vs mice fed with regular chow; ## P < 0.01 vs mice fed with HFD). (b) Representative pictures of H&E staining for adipose tissues (Arrowheads points to crown-like structures). (c) Results of quantitative analysis (** P < 0.01 vs mice fed with regular chow; ## P < 0.01 vs mice fed with HFD).

Full size image

Rutin Suppressed the Transcription of Genes Involved in Chronic Inflammation in WAT

To explore the possible mechanism, we measured the mRNA levels of genes that are pivotal for chronic inflammation in WAT. HFD-feeding dramatically increased the expression of F4/80, Cd11c and Cd68 by ~29.2-fold, ~26.3-fold and ~15.4-fold, respectively (Fig. 6a). Rutin completely blocked the elevated transcription of F4/80 and Cd11c, and greatly reduced the mRNA level of Cd68 by 78.4% (Fig. 6a). Interestingly, HFD-feeding also increased the mRNA levels of Cd206 and Arg1, two marker genes for alternatively activated macrophages, by ~3.8-fold and ~1.9-fold, respectively (Fig. 6b). However, rutin partly repressed Cd206 and increased Arg1 by ~7.5-fold (Fig. 6b). Moreover, HFD-feeding elevated the mRNA levels of Tnfα and Mcp1 in WAT (Fig. 6c). Rutin greatly repressed these elevations (Fig. 6c). Additionally, neither HFD-feeding nor rutin treatment significantly changed the transcription levels of genes involved in energy expenditure in WAT, including Pgc1α, Pgc1β, Ucp1, Ucp2 and Ucp3 (Fig. 6d).

Fig. 6
figure 6

Rutin-mediated transcription inhibition of the genes involved in chronic inflammation in WAT of mice fed a HFD. (a) Relative mRNA levels of marker genes for classically activated macrophages (n = 4, ** P < 0.01 vs mice fed with regular chow; ## P < 0.01 vs mice fed with HFD). (b) Relative mRNA levels of marker genes for alternatively activated macrophages (n = 4, ** P < 0.01 vs mice fed with regular chow; ## P < 0.01 vs mice fed with HFD). (c) Relative mRNA levels of pro-inflammatory cytokines (n = 4, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed with HFD). (d) Relative mRNA level of genes involved in energy metabolism.

Full size image

Rutin Up-Regulated the Transcription of Genes Involved in Energy Expenditure in BAT

In BAT, rutin significantly increased the mRNA levels of Dio2 and Elovl3 by ~6.9-fold and ~5.1-fold, respectively (Fig. 7a). Moreover, rutin also increased the transcription of Pgc1α by ~1.9-fold (Fig. 7b). Additionally, we determined the mRNA levels of other genes involved in energy expenditure and nutrient metabolism in BAT, including Ucp1, Ucp2, Ucp3, Pgc1β, Cpt1α, Cpt1β, Pdk4 and Glut4. No mRNA levels of these genes were significantly changed by HFD-feeding or rutin treatment (Fig. 7c and d)

Fig. 7
figure 7

Rutin-mediated modulation of gene expression in BAT of mice fed a HFD. (a) Relative mRNA level of BAT signature genes (n = 4, ** P < 0.01 vs mice fed with regular chow; ## P < 0.01 vs mice fed with HFD). (b–c) Relative mRNA level of genes involved in energy expenditure (n = 4, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed a HFD). (d) Relative mRNA level of genes involving glucose and lipids metabolism.

Full size image

Rutin Blocked HFD-Induced Fatty Liver

HFD feeding for 8 weeks markedly increased the weight of livers by ~31.3%, and rutin completely blocked the increase (Fig. 8a). Consistently, HFD-feeding caused apparent fatty liver, as evidenced by the vacuoles in H&E staining and further confirmed by Oil-red O staining, and rutin completely blocked the development of HFD-induced fatty liver (Fig. 8b). Quantitative determination of triglycerides in the liver leads to the same conclusion (Fig. 8c). Both AST and ALT levels were in normal ranges for all animals fed with either HFD or regular chow (Fig. 8d).

Fig. 8
figure 8

Rutin-mediated blockade of HFD-induced fatty liver. (a) Liver weight (n = 5, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed a HFD). (b) Representative pictures of H&E staining and Oil-red O staining. (c) Liver triglyceride levels (n = 5, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed a HFD). (d) Blood level of AST and ALT.

Full size image

Rutin Modulated the Transcription of Genes Involved in Inflammation and Nutrient Metabolism in the Liver

Results in Fig. 9a indicate a slight increase in mRNA levels of several inflammatory genes including F4/80, Tnfα and Ifnγ in animals fed a HFD, but the difference was not statistically significant. Treatment with rutin did not show a notable difference. However, HFD-feeding increased the mRNA level of Srebp1c by ~2.8-fold, but had no significant impact on Acc, Fas and Scd1 (Fig. 9b). Rutin suppressed the elevated transcription of Srebp1c by ~78.9% (Fig. 9b). Additionally, rutin increased the transcription of Pdk4 and decreased the HFD-induced transcription increase of Cd36, Pepck and G6p in the livers (Fig. 9c).

Fig. 9
figure 9

Effect of rutin on hepatic gene expression (a) Relative mRNA levels of genes involving inflammation (## P < 0.01 vs mice fed a HFD). (b) Relative mRNA levels of genes for lipogenesis (n = 4, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed a HFD). (c) Relative mRNA levels of genes for glucose and lipids metabolism (n = 4, ** P < 0.01 vs mice fed with regular chow; ## P < 0.01 vs mice fed with HFD).

Full size image

Rutin Maintained Glucose Sensitivity in Mice fed a HFD

Mice fed a HFD for 8 weeks showed significantly impaired glucose sensitivity, and treatment with rutin maintained such sensitivity (Fig. 10a). AUC calculations make this point clearer (Fig. 10b). To assess insulin sensitivity in these mice, we performed ITT. HFD-feeding caused an apparent insulin resistance, and treatment with rutin completely blocked this resistance (Fig. 10c). Blood insulin determinations show that HFD-feeding increased the levels of insulin by ~4.6-fold and treatment with rutin greatly reduced the elevation by ~71.2% (Fig. 10d).

Fig. 10
figure 10

Rutin-mediated inhibition of HFD-induced glucose tolerance and insulin resistance. (a) Glucose profiles of IPGTT (n = 5, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed a HFD). (b) Results of AUC calculations (n = 5, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed a HFD). (c) Results of ITT (n = 5, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed a HFD). (d) Insulin level in blood (n = 5, ** P < 0.01 vs mice fed a regular chow; ## P < 0.01 vs mice fed a HFD).

Full size image

Discussion

In this study, we demonstrate that rutin suppresses palmitic acid-triggered inflammation in macrophages (Fig. 1) and blocks HFD-induced obesity in mice (Fig. 4). The anti-inflammatory effect of rutin in macrophages is correlated with its activity in suppressing ER stress and ROS production (Figs. 2 and 3). The anti-obese effect of rutin in HFD-feeding mice was associated with its action in blocking macrophage infiltration (Fig. 5) and chronic inflammation in adipose tissue (Fig. 6).

Recent studies by Henao-Mejia et al. and Stienstra et al. show that chronic inflammation plays critical roles in the pathogenesis of obesity and its complications (5,6). Chronic inflammation in obesity is primarily mediated by macrophages (11,12). Emerging evidence shows that macrophages can be activated by saturated fatty acids (13,14). Moreover, recent studies by Cruz et al. and Bulua et al. demonstrate that the promoted inflammation is correlated with increased ROS production (15,16). In addition, recent studies also show that ER stress may play important roles in promoting inflammation as well (17,18). Our results are in concurrence with these earlier studies in support that rutin suppresses the macrophage activation by decreasing ROS production and ER stress (Figs. 2 and 3).

Macrophage infiltration into adipose tissue is one typical feature of obesity (19,20). The original observation was reported by Weisberg et al. (19) and Xu et al. (20), and subsequently confirmed and expanded by others using different models (21–23). Although the detailed molecular mechanism for this process needs additional investigation, it is largely accepted that hypertrophy and necrosis of adipocytes play critical roles (24). Hypertrophy is a central cause for adult obesity and is usually associated with adipocyte death (19,24). Moreover, Cinti and colleagues proposed that necrosis of adipocytes, driven by hypertrophy, is one of the predominate signals regulating macrophage infiltration (21,25). The infiltrated macrophages aggregate around necrotic adipocytes, leading to development of crown-like structures and chronic inflammation (21,25). Consistent with these studies, our data show that HFD-feeding causes apparent hypertrophy of adipocytes, resulting in an increased number of crown-like structures and elevated chronic inflammation in adipose tissue (Figs. 5 and 6). Treatment with rutin significantly suppressed this process, suggesting that the anti-obese effect of this anti-oxidant may be achieved, at least in part, through blocking necrosis and chronic inflammation in adipose tissues. Additionally, rutin also has a direct effect on hepatocytes and adipocytes. A previous study by Wu et al. using hepatocarcinoma cells demonstrated that rutin can inhibit oleic acid-induced lipid accumulation in hepatocytes in vitro, probably via reducing lipogenesis and oxidative stress (26). In addition, using 3 T3-L1 cells Choi et al. demonstrated that rutin is capable of suppressing differentiation of pre-adipocytes in vitro (27). These direct effects may be related to the anti-oxidant activity of rutin.

Insulin resistance is a major complication associated with obesity, and its underlying mechanism remains unclear (28,29). Shulman and colleagues proposed that ectopic accumulation of lipids and dysregulation of intracellular signal pathways may play central roles in the etiology of insulin resistance (30,31). On the other hand, accumulating evidence links chronic inflammation to the induction of insulin resistance, suggesting that macrophage-related inflammation may also contribute to the pathogenesis of obesity-induced insulin resistance (20, 32,33). Our data show that treatment with rutin blocks development of insulin resistance and maintains glucose sensitivity in mice fed a HFD (Fig. 10). These effects were correlated with the blocked ectopic fat accumulation in livers and the suppressed chronic inflammation in adipose tissues (Figs. 6 and 8).

As a potent natural anti-oxidant, rutin can be found in many fruits and vegetables. Previous studies have suggested that rutin be used as a dietary supplement to modulate blood level of lipids and glucose. Additionally, it has also been applied in clinic for treatment of many diseases including poor circulation, high blood pressure and capillary fragility (34). The underlying mechanism, however, was considered complicated because no specific target has been identified. Our current study shows that rutin blocks macrophage activation and represses chronic inflammation in adipose tissue, providing new information for better understanding its beneficial effects and for using rutin to manage metabolic disorders. Evidently, further studies at molecular levels are still needed to see whether other pathways are also involved.

Rutin altered the expression levels of multiple genes in mice fed a HFD. Arg1 is a marker for alternatively activated macrophages (M2 macrophages), and elevated expression of Arg1 is usually associated with a higher ratio of M2 macrophages that are essential to maintain the homeostasis of adipose tissue (35). Pgc1α, Dio2 and Elovl3 are three critical genes tightly involved in energy expenditure in BAT (36). Rutin up-regulated expression levels of these three genes, indicating that this natural anti-oxidant may be able to increase energy expenditure. Pdk4 encodes pyruvate dehydrogenase kinase isozyme 4 which is an enzyme located in the matrix of the mitochondria and regulates the balance between glucose and lipid metabolism (37). Rutin increased transcription of Pdk4 in liver, suggesting that it may elevate lipid oxidation in this organ. Further studies are warranted to elucidate how rutin treatment altered expression levels of these genes in vivo.

In conclusion, our results show that rutin is able to block HFD-induced obesity, fatty liver and insulin resistance in mice, and these beneficial effects were correlated with a blockade of macrophage infiltration and chronic inflammation in adipose tissue. Our results confirm that supplementation of rutin is a promising strategy for preventing HFD-induced obesity and its associated complications.