Dietary Phaseolus vulgaris extract alleviated diet-induced obesity, insulin resistance and hepatic steatosis and alters gut microbiota composition in mice

Journal of Functional Foods 20 (2016) 236–244

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Dietary Phaseolus vulgaris extract alleviated diet-induced obesity, insulin resistance and hepatic steatosis and alters gut microbiota composition in mice Haizhao Song a,b, Wen Han a, Fujie Yan a, Dongdong Xu a, Qiang Chu a, Xiaodong Zheng a,b,* a b

College of Biosystems Engineering and Food Science, Zhejiang university, Hangzhou, China Fuli Institute of Food Science, Zhejiang University, Hangzhou, China

A R T I C L E

I N F O

A B S T R A C T

Article history:

Phaseolus vulgaris may reduce appetite, body weight and gut microbiota and has been shown

Received 13 August 2015

to contribute to the development of obesity, type 2 diabetes and non-alcoholic fatty liver

Received in revised form 7 October

disease. This study was conducted to investigate the impact of P. vulgaris extract (PVE) on

2015

obesity and obesity-associated metabolic syndromes, and determine whether the protec-

Accepted 20 October 2015

tive effects of PVE were associated with modulation of gut microbiota. C57BL/6J mice were

Available online

fed low-fat diet, high-fat diet or high-fat diet supplemented with PVE of 50 mg/kg. The composition of gut microbiota was analysed by 16S rDNA sequencing. Our results showed that

Keywords:

PVE treatment reduced body weight and improved hepatic steatosis and insulin resistance

Phaseolus vulgaris extract

in high-fat diet-fed mice. PVE administration increased the relative abundance of

Obesity

Bifidobacterium, Lactobacillus and Akkermansia at the genus level. In conclusion, PVE pro-

Insulin resistance

tects from diet-induced obesity, insulin resistance and hepatic steatosis, and these beneficial

Hepatic steatosis

effects were associated with the PVE-related modulation of gut microbiota.

Gut microbiota

1.

Introduction

Obesity increases the risk of various metabolic diseases, such as insulin resistance, type 2 diabetes, dyslipidaemia, hepatic steatosis, hypertension and cardiovascular diseases (Everard & Cani, 2013; Popkin, Kim, Rusev, Du, & Zizza, 2006). As a major public health problem, obesity has become a focus of research in recent years, and a growing body of literature supports

© 2015 Elsevier Ltd. All rights reserved.

that dietary modifications, including plant-derived foods consumption, offer a promising therapy to ameliorate obesity and its complications (Baboota et al., 2013; Dembinska-Kiec, Mykkanen, Kiec-Wilk, & Mykkanen, 2008; Espin, Garcia-Conesa, & Tomas-Barberan, 2007; Liu, 2003; Zang, Shimada, Kawajiri, Tanaka, & Nishimura, 2014). As known, at least 1014 bacteria consisting of more than 2000 species exist in the human gastrointestinal tract, exerting a distinct influence on host metabolism, nutrient digestion, energy

* Corresponding author. College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road, Xihu District, Hangzhou, 310058, China. Tel.: +86 571 86971139; fax: +86 571 86971139. E-mail address: [email protected] (X. Zheng). Chemical compounds studied in this article: Phaseolamin (PubChem CID: 91572); Chloroform (PubChem CID: 6212); Methanol (PubChem CID: 887); Glucose (PubChem CID: 5793); Haematoxylin (PubChem CID: 442514); Eosin yellowish (PubChem CID: 11048); Formaldehyde (PubChem CID: 91572); Dimethylbenzene (PubChem CID: 7259); Ethanol (PubChem CID: 702); Isopropanol (PubChem CID: 3776). http://dx.doi.org/10.1016/j.jff.2015.10.022 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 20 (2016) 236–244

utilization and storage (Xu & Gordon, 2003; Xu et al., 2007). So abnormal changes of the gut microbiota could lead to undesirable impacts on the host’s health, for instance, contributing to the development of obesity, type 2 diabetes and nonalcoholic fatty liver disease (Everard & Cani, 2013; Fei & Zhao, 2013; Kovatcheva-Datchary & Arora, 2013; Zhao, 2013). Highfat diet has been shown to reshape the gut microbiota composition, especially altering the proportion of the Bacteroidetes and Firmicutes, contributing to the development of obesity and other metabolic disorders (Everard et al., 2011; Hildebrandt et al., 2009). Likewise, modified gut microbiota could also alleviate obesity and obesity-related complications. For example, decrease in the ratio of Bacteroidetes to Firmicutes and increase of the proportion of Akkermansia could alleviate dietinduced obesity and type 2 diabetes (Anhe et al., 2015; Shin et al., 2014). A growing body of literature proves that the modulation of gut microbiota by dietary functional food ingredients has recently become a major topic of research in biology and functional foods research (Baboota et al., 2013; Guida & Venema, 2015). Therefore, it is important to understand the interaction between gut microbiota and functional foods and explore safe and effective therapeutic strategies to ameliorate obesity and other metabolic diseases. Phaseolus vulgaris, also known as white kidney bean, is a popular food consumed mostly in Asia and Eastern countries. A growing body of laboratory animal studies indicate that acute or chronic administration of the derivatives, extracts, ingredients of P. vulgaris significantly reduce appetite, food intake, carbohydrate absorption and metabolism, lipid accumulation, body weight gain, glycaemia and glucose absorption in lean and obese animals (Fantini et al., 2009; Maccioni et al., 2010; Pusztai, Bardocz, & Ewen, 2008; Pusztai et al., 1998). A series of clinical trials also support that P. vulgaris may constitute a potentially safe and effective remedy for the treatment of obesity and its associated metabolic syndromes (Boivin, Zinsmeister, Go, & Dimagno, 1987; Celleno, Tolaini, D’Amore, Perricone, & Preuss, 2007; Layer, Carlson, & Dimagno, 1985; Layer, Zinsmeister, & Dimagno, 1986; Udani & Singh, 2007). Two potential molecular mechanisms underlying the reducing effect of P. vulgaris on food intake and body weight have been proposed: (a) The presence of α-amylase inhibitors results in a suppression of starch metabolism and a reduced feed efficiency (Tormo, Gil-Exojo, Romero de Tejada, & Campillo, 2004, 2006); (b) The phytohaemoagglutinin, a lectin rich in P. vulgaris, could modulate the activity of glucagon-like peptides and cholecystokinin by binding to the epithelium of stomach and the brush border of small intestine, colon and cecum, which play an important role in the digestive process and appetite control (Baintner, Kiss, Pfuller, Bardocz, & Pusztai, 2003; Pusztai et al., 1998). Considering the beneficial effects of P. vulgaris on human health and its long-period food culture, we postulated that P. vulgaris might also exert its beneficial effect through the modulation of gut microbiota besides the above two mechanisms of action. In addition, the glucose homeostasis-improving effect of P. vulgaris has not been fully elucidated and the influence of P. vulgaris on insulin also receives little attention. Moreover the anti-hepatosteatosis effect of P. vulgaris has not been reported yet. So the objective of the present study was to evaluate the influence of PVE on obesity and obesityassociated insulin resistance and hepatic steatosis in high-fat

237

diet-fed mice and determine whether the beneficial effects of PVE are associated with the modulation of gut microbiota. To the best of our knowledge, this is the first study to investigate the interrelationship between gut microbiota and PVEproduced beneficial effects.

2.

Materials and methods

2.1.

Chemical reagents

Phase 2, a standardized P. vulgaris extract containing 3% phaseolamin, was supplied by ZeLang (Nanjing, China). The solvents and chemicals were obtained from Aladdin (Shanghai, China).

2.2.

Animals and diets

All the experimental protocols in this research were approved by the Committee on the Ethics of Animal Experiments of Zhejiang University (Permission Number: ZJU201550501). Male C57BL/6J mice (4 weeks old, n = 48; the National Breeder Center of Rodents, Shanghai, China) were maintained, four animals per cage, in an environment controlled at 23 ± 3 °C and 12hour light/dark, with water and food ad libitum. After one week of acclimation, the mice were randomly divided into the following three groups (n = 12): LFD, mice fed low-fat diet; HFD, mice fed high-fat diet with free accesses to water; PVE, mice fed high-fat diet with supplementation of P. vulgaris extract of 50 mg/kg. The ingredients and energy densities of the diets are listed in the Supplementary Table S1. The body weights and intakes of mice were monitored once a week. After 14 weeks of treatment, the mice were fasted for 12 h and then sacrificed by decapitation. Blood was drawn in tubes and centrifuged at 2000 g for 15 min to collect serum samples. The hearts, livers, kidneys, spleens, epididymal and perirenal white adipose and interscapular brown adipose were collected and weighted.

2.3.

Biochemical analysis

Concentrations of serum triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glucose were measured using an automatic biochemistry analyser (ACCUTE TBA40FR, Japan) according to the manufacturer’s instructions, and the serum levels of insulin and adiponectin were determined by enzymatic methods using commercial kits (R&D Systems, Minneapolis, MN, USA). Liver tissues were homogenized and the total lipids were extracted using chloroform/methanol (v/v, 1:2) following the previously described method (Folch, Lees, & Sloane Stanley, 1957). The liver TG and TC content in the lipid extracts were measured by commercially available kits (Elabscience) according to the manufacturer’s instructions.

2.4.

Glucose homeostasis

After 12 weeks of treatment, mice were fasted overnight and an oral glucose tolerance test (OGTT) was conducted after oral

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gavage of glucose (2 g/kg). Then the concentrations of blood glucose were measured at 0, 30, 60, 90 and 120 min, respectively, using an Accu-Check glucometer (Roche, Mannheim, Germany). At the end of the experiment, the homeostasis model assessment of insulin sensitive index (HOMA-IS) and insulin resistance (HOMA-IR) were calculated based on the serum concentrations of insulin and glucose as follows:

HOMA-IR = serum glucose ( mmol L ) × serum insulin ( mU ) 22.5 HOMA-IS = 1 [serum glucose ( mmol L ) × serum insulin ( mU )]

2.5.

Histology analysis

Liver and epididymal white adipose tissues were fixed in 10% buffered formalin, then paraffin-embedded, cut into 6 µm sections and stained with haematoxylin and eosin (H&E). For oil red O staining, liver tissues were embedded in Optimal Cutting Temperature gel, and the air-dried 5 µm thick sections got dipped in formalin and washed with 0.5% oil red O solution.The images were used to analyse the hepatic lipid accumulation and white adipocyte size, and the steatosis grades were scored using the previously described method (Kleiner et al, 2005).

2.6.

Gut microbiota analysis

The methods used in bacterial DNA extraction, 16S rDNA gene sequencing and gut microbiota analysis were described in the online supplementary methods.

2.7.

Items

LFD

HFD

PVE

Initial BW (g) 18.26 ± 0.15 18.62 ± 0.28 18.29 ± 0.29 45.47 ± 1.01b 38.25 ± 0.71c Final BW (g) 29.18 ± 0.62a Cumulative weight 10.63 ± 0.73a 23.99 ± 1.13b 17.84 ± 0.73c gain (g) Food intake 3.16 ± 0.1a 2.8 ± 0.07b 2.51 ± 0.08c (g/mouse/day) Calorie intake 12.17 ± 0.39a 13.26 ± 0.33b 11.84 ± 0.37a (kcal/mouse/day) Tissue index (% BW) 0.32 ± 0.02b 0.36 ± 0.02b Heart 0.51 ± 0.03a a b Liver 4.14 ± 0.14 2.66 ± 0.16 3.03 ± 0.18b Spleen 0.22 ± 0.01a 0.16 ± 0.01b 0.17 ± 0.02b a b Kidney 1.23 ± 0.04 0.85 ± 0.02 0.98 ± 0.04c Pancreas 0.41 ± 0.02a 0.28 ± 0.02b 0.33 ± 0.01c a b Epididymal WAT 1.7 ± 0.2 5.54 ± 0.26 3.74 ± 0.22c Interscapular BAT 0.59 ± 0.2 0.31 ± 0.04 0.29 ± 0.02 Serum parameters 1.36 ± 0.08b 1.11 ± 0.07a TG (mmol/L) 0.96 ± 0.07a a b TC (mmol/L) 3.45 ± 0.17 5.9 ± 0.17 4.19 ± 0.18c HDL-C (mmol/L) 1.76 ± 0.07a 2.16 ± 0.06b 2.41 ± 0.1c a b LDL-C (mmol/L) 0.32 ± 0.04 0.59 ± 0.03 0.45 ± 0.02a TNFα (U/L) 480.05 ± 17.39 535.49 ± 29.7 459.58 ± 29.38 76.53 ± 5.32b 91.42 ± 3.18a Adiponectin (µg/L) 99.17 ± 5.18a LFD, group fed with low fat diet; HFD, group fed with high-fat diet; PVE, group fed with high-fat diet supplemented with Phaseolus vulgaris extracts at 200 mg/kg. BW, body weight; WAT, white adipose; BAT, brown adipose; TG, triacylglycerol; TC, total cholesterol; HDL-C, highdensity lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. Values are presented as means ± SEM (n = 12). Means not sharing a common superscript differ significantly among treatments (P < 0.05, ANOVA).

Statistical analysis

Data are presented as mean ± SEM. SPSS 19.0 statistical software was used to perform the statistical analyses, and the differences among the different dietary groups were assessed by one-way analysis of variance (ANOVA) followed by Duncan’s post hoc test. p < 0.05 was considered statistically significant.

3.

Table 1 – Tissue weights and serum parameters in mice.

Results

3.1. PVE reduced food intake and body weight and improved serum lipid profile PVE supplementation significantly reduced high-fat dietinduced body weight gain as well as the relative weight of visceral white adipose and resulted in a lower final body weight compared to mice fed high-fat diet alone (Table 1). High-fat diet also induced a decrease in the relative weight of heart, liver, pancreas, spleen and kidney while PVE administration tended to reverse the trend. And mice in HFD group consumed less food and calorie than those in LFD group, and PVE administration further resulted in a reduction in food and calorie intake (Table 1). In addition, high-fat diet feeding induced an increase in the serum levels of TG, TC, LDL-C and HDL-C, while PVE administration resulted in a significant decrease in serum concentrations of TG, TC, LDL-C but not HDL-C. Moreover, PVE

supplementation significantly increased the serum level of adiponectin and tended to decrease the concentration of TNFα (Table 1).

3.2. PVE administration attenuated diet-induced hepatic steatosis and adipose tissue hypertrophy PVE supplementation alleviated high-fat diet-induced lipid accumulation and formation of steatosis in the liver (Fig. 1A, B), and resulted in a significantly lower hepatic TG and TC levels and serum ALT and AST levels (Fig. 1E,F,G,H). The antihepatosteatosis effect of PVE was also confirmed by the lower steatosis grade compared to that in mice fed high-fat diet alone (Fig. 1D). In addition, H&E staining revealed that PVE administration reduced the cell size of white adipocyte, suggesting the alleviating effect of PVE on high-fat diet-induced adipose hypertrophy (Fig. 1C).

3.3. PVE administration improved diet-induced glucose intolerance and insulin resistance High-fat diet feeding induced an elevation of fasting serum levels of glucose and insulin accompanied by an increased HOMA-IR and decreased HOMA-IS index. PVE administration resulted in a significant reduction in the serum glucose and

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239

Fig. 1 – Phaseolus vulgaris extract (PVE) supplement attenuates high-fat diet-induced hepatic steatosis and adipocyte hypertrophy. H&E staining (A) and oil red O staining (B) of liver. (C) H&E staining of epididymal white adipose (eWAT). (D) Hepatic steatosis grade. Hepatic triacylglycerols (E) and cholesterol levels (F). Serum concentrations of ALT (G) and AST (H). LFD, mice fed low-fat diet; HFD, mice fed high-fat diet; PVE, mice fed high-fat diet with supplementation of PVE. n = 6 (D), n = 12 (E, F, G and H). Values are presented as mean ± SEM. Data not sharing a common superscript differ significantly among groups (P < 0.05, ANOVA).

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resulted in a decrease in the relative abundance of Firmicutes but a significant increase in Verrucomicrobia and Actinobacteria compared to mice fed high-fat diet alone at the phylum level (Fig. 3B). Moreover, as shown in Fig. 3C, PVE administration significantly increased the relative abundance of Bifidobacterium, Lactobacillus and Akkermansia at the genus level.

4.

Fig. 2 – Phaseolus vulgaris extract (PVE) administration improves glucose tolerance, insulin resistance and insulin sensitivity. The fasting serum levels of glucose (A) and insulin (B). (C) Oral glucose tolerance test (OGTT). (D) HOMA-IR index. (E) HOMA-IS index. LFD, mice fed low-fat diet; HFD, mice fed high-fat diet; PVE, mice fed high-fat diet with supplementation of PVE. n = 12 (A), n = 10 (B, D and E), n = 8 (C). Values are presented as mean ± SEM. Data not sharing a common superscript differ significantly among groups (P < 0.05, ANOVA).

insulin levels along with a decrease in HOMA-IR index but an increase in HOMA-IS index (Fig. 2A,B,D,E). Consistent with these results, the OGTT test revealed a lower glucose levels in mice supplemented with PVE than that fed high-fat diet alone (Fig. 2C). These data suggested that PVE administration could improve glucose tolerance, insulin resistance and insulin sensitivity in diet-induced obese mice.

3.4.

PVE administration modified gut microbiota

Principal component analysis (PCA) was used to cluster bacterial communities based on the different diet/treatment groups at the genus level. The PCA result revealed that the three samples formed distinct clusters in the ordination plot, suggesting that different diet/treatment induced the main alternations in the gut microbiota of mice (Fig. 3A). In addition, high-fat diet induced a significant increase in the proportion of Firmicutes but a decrease in Bacteroidetes, Proteobacteria and Verrucomicrobia, while PVE administration

Discussion

Dietary interventions have become an important strategy for preventing obesity and obesity-associated complications. P. vulgaris has been reported to possess the pharmacological effects on appetite, overweight, hyperlipidaemia and hyperglycaemia, depending on its starch blocker activity attributed to the presence of a-amylase inhibitors and phytohaemagglutinin (Boivin et al., 1987; Carai et al., 2009; Celleno et al., 2007; Everard et al., 2011; Fantini et al., 2009; Layer et al., 1985, 1986; Pusztai et al., 1998, 2008; Udani & Singh, 2007). Considering that NAFLD is a main complication of obesity and gut microbiota plays a curial role in the development of obesity, and the influence of PVE on NAFLD and gut microbiota has not been reported, so the present study was conducted to systemically investigate the anti-obesity, anti-hyperglycaemic and anti-NAFLD effects of PVE and determine whether the gut microbiota contributed to the beneficial effects of PVE. Our results clearly indicated that PVE administration significantly reduced food and calorie intake, decreased high-fat diet-induced body weight gain, and improved lipid profile, hepatic steatosis and insulin resistance in mice. Moreover the beneficial effects of PVE were associated with modulations of gut microbiota. A growing body of experimental evidence has proved the reducing effects of P. vulgaris and its extracts on appetite, food intake, body weight, lipid accumulation and glycaemia (Boivin et al., 1987; Carai et al., 2009; Layer et al., 1985, 1986; Pusztai et al., 1998, 2008; Udani & Singh, 2007). Consistent with the previous studies, our results indicated that oral supplementation of PVE at 50 mg/kg for 14 weeks resulted in a significant reduction in food and calorie intake and body weight gain. PVE administration also induced a significant decrease in serum concentrations of TG, TC, LDL-C and glucose. Unlike the previous studies, our results revealed that PVE administration also significantly downsized the adipocyte of the epididymal white adipose, indicating that PVE supplementation could alleviate high-fat diet-induced adipose tissue hypertrophy in mice. Hepatic lipid accumulation is a sign of liver steatosis which, combined with inflammation, oxidative stress and mitochondrial dysfunction, can lead to the occurrence and aggregation of NAFLD (Charlton, 2004). Until now, no evidence has identified the impact of PVE on NAFLD. In our study, H&E staining of livers indicated that PVE supplementation significantly reduced high-fat diet-induced hepatic lipid accumulation, attenuated the swelling of hepatocytes and alleviated the formation of liver steatosis. In combination with the results that PVE administration significantly lowered steatosis grades, hepatic TG and TC and serum ALT and AST levels, we believed that dietary supplementation with PVE could significantly alleviate high-fat diet-induced hepatic steatosis in mice. To the best of our knowledge, this is the first report demonstrating that PVE possessed anti-hepatosteatosis effect.

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Fig. 3 – Phaseolus vulgaris extract (PVE) administration alters the composition of gut microbiota. (A) Principal component analysis (PCA) scores plot of gut microbiota at the genus level. (B) The composition and proportion of bacterial communities at the phylum level. (C) Heat map analysis showing the changes in the relative abundance of each identified genus. For an individual genus, the colour intensity is normalized to represent its relative ratio in the three samples. LFD, mice fed low-fat diet; HFD, mice fed high-fat diet; PVE, mice fed high-fat diet with supplementation of PVE. (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

Obesity is often accompanied by insulin resistance, thus increasing the risk of type II diabetes (Kruszynska & Olefsky, 1996). Though the reducing effect of P. vulgaris on glycaemia has been reported (Rondanelli, Giacosa, Orsini, Opizzi, & Villani, 2011; Spadafranca et al., 2013), the glucose homeostasis-improving effect of PVE has not been fully elucidated and the influence of P. vulgaris on insulin also received little attention. On the one hand, consistent with the previous studies, we found that PVE supplementation significantly reduced the fasting serum glucose levels. Moreover, the OGTT test showed a progressively reduced glucose levels in mice supplemented with PVE after glucose injection, suggesting an improved glucose tolerance. On the other hand, our results indicated that PVE administration significantly reduced the fasting serum insulin level in high-fat

diet-fed mice, and the hyperinsulinemia-improving effect was also supported by the decreased HOMA-IR index, suggesting the improving effect of PVE on insulin resistance. Moreover, the increased HOMA-IS index suggested that PVE administration improved the insulin sensitivity in mice. In addition, we observed that PVE administration increased the serum levels of adiponectin, which played a crucial role in fatty acid oxidation and glucose metabolism (Xu & Gordon, 2003). This finding might suggest one potential action site of PVE. There is a high content of phaseolin, a classical α-amylase inhibitor, in the P. vulgaris extract. As known, α-amylase is an important enzyme that catalyzes the hydrolysis of α-(1,4)glycosidic bonds of starch polymers, and various other enzymes present in the brush border of the small intestine convert the

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produced oligosaccharides to monosaccharides that can be absorbed. Inhibition of α-amylase could suppress the starch metabolism and appetite, delay gastric emptying, thus resulting in a decrease in food intake and body weight, in turn, a decrease in glycaemia (Tormo et al., 2004, 2006). Moreover, the undigested starches have similar effects with the resistant starches, which can be fermented by the colonic bacteria to produce carbon dioxide, short-chain fatty acids (SCFA) and methane, and influence the intestinal ecosystem of host (Haenen et al., 2013). In addition, the extract used in our study (Phase 2) is a standardized white kidney bean extract prepared by heated processing conditions to substantially inactivate the activity of phytohaemoagglutinin while preserving the α-amylase inhibiting ability. So we believe that the mechanism behind the improving effects of PVE relies on the activity of the a-amylase-inhibiting activity of phaseolin. Obesity is a consequence of gut flora and diet interaction, and growing evidence has confirmed that gut microbiota, which is involved in the control of energy homeostasis and body weight, plays a crucial role in the management of obesity and type II diabetes (Fei & Zhao, 2013; Guida & Venema, 2015; Kotzampassi, Giamarellos-Bourboulis, & Stavrou, 2014; Kovatcheva-Datchary & Arora, 2013; X. Zhang et al., 2015; Zhao, 2013). Because of the long-period food culture of P. vulgaris and its beneficial effects on obesity, the investigation of the influence of PVE on gut microbiota would be very helpful to understand the mechanism by which PVE exerted its antiobesity and anti-diabetic effect. Our results showed that highfat diet feeding and PVE treatment promoted the main alternations of the gut microbiota in mice. Growing evidence has shown that Firmicutes and Bacteroidetes, the majority bacterial phyla of the intestinal microbiome, play a crucial role in the regulation of host energy homeostasis (Cani et al., 2007; Hildebrandt et al., 2009), and in this study, analysis of faecal samples did reveal an obvious link between obesity and the increase in Firmicutes and drop in Bacteroidetes at the phylum level, which was consistent with the previous studies. At the phylum level, PVE administration decreased the proportion of Firmicutes and increased the proportion of Verrucomicrobia and Actinobacteria, and no significant change was observed in the relative proportion of Firmicutes to Bacteroidetes compared to mice fed high-fat diet alone. Zhang et al. (2009) reported that Verrucomicrobia was significantly enriched after gastric bypass, suggesting a positive link between appetite control and Verrucomicrobia. Actinobacteria phyla has been proved to conserve the bile salt hydrolase, which could improve the mucosal defences, decrease cholesterol levels and regulate lipid metabolism of the host (Jones, Begley, Hill, Gahan, & Marchesi, 2008; Mai, 2004). This result suggested that Verrucomicrobia and Actinobacteria might be one potential action site for PVE to exert its reducing effects on weight gain and hyperlipidaemia. Besides the significant changes of the bacterial communities at the phylum level, our results also revealed that PVE administration significantly increased the relative abundance of Bifidobacterium, Lactobacillus and Akkermansia at the genus level. Santacruz et al. found that weight loss was closely associated with an increased proportion of Lactobacillus (Santacruz et al., 2009). Several reports have also shown that high-fat diets notably decreases the proportion of the Gram-positive Bifidobacteria, and the increased Bifidobacterium

species in intestinal microbiota profile may be protective against obesity and weight gain (Duncan et al., 2007; Kalliomaki, Collado, Salminen, & Isolauri, 2008). Moreover, a study reported that increased proportion of Bifidobacteria in gut microflora could improve high-fat diet-induced diabetes in mice (Cani et al., 2007). More importantly, we found that the beneficial effects of PVE on metabolic disorders were associated with a robust increase in the proportion of Akkermansia. Previous studies have shown that tea polyphenols and cranberry extract could induce an increased Akkermansia abundance, combating obesity and type 2 diabetes (Anhe et al., 2015; Axling et al., 2012; Kemperman et al., 2013), and oral administration of Akkermansia significantly improves high-fat dietinduced metabolic diseases and Akkermansia administration could mimic the anti-diabetic effects of metformin in diabetic mice (Liou et al., 2013; Shin et al., 2014). Along with these literature, our results indicated that PVE induced enrichment in Bifidobacterium, Lactobacillus and Akkermansia might be sufficient to reverse high-fat diet metabolic syndromes, including obesity, hyperglycaemia, insulin resistance and hepatic steatosis, without a major modification in the relative proportion of Firmicutes to Bacteroidetes. In summary, our results not only verified the previous findings that P. vulgaris treatment reduced appetite and body weight but also demonstrated the improving effect of Phaseolus vulgaris on hepatic steatosis and insulin resistance, implying the potential dietary choice of Phaseolus vulgaris as a functional food ingredient for the treatment of obesity, type 2 diabetes, nonalcoholic fatty liver diseases (NAFLD) and other related metabolic syndromes. This is the first study proposing that Phaseolus vulgaris might prevent obesity and its related metabolic disorders through a beneficial influence on the gut microbiota.

Acknowledgments This work was supported by the National Key Technology R&D Program of China (Grant No. 2012BAD33B08), Zhejiang Provincial Natural Science Foundation of China (Grant No. Z14C200006) and the Foundation of Fuli Institute of Food Science, Zhejiang University (Grant No. KY201301).

Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.jff.2015.10.022.

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