Purified Betacyanins from Hylocereus undatus Peel Ameliorate

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Purified Betacyanins from Hylocereus undatus Peel Ameliorate Obesity and Insulin Resistance in High-Fat-Diet-Fed Mice Haizhao Song,†,‡ Qiang Chu,† Dongdong Xu,† Yang Xu,† and Xiaodong Zheng*,†,‡ †

College of Biosystems Engineering and Food Science, and ‡Fuli Institute of Food Science, Zhejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China S Supporting Information *

ABSTRACT: Natural bioactive compounds in food have been shown to be beneficial in preventing the development of obesity, diabetes, and other metabolic diseases. Increasing evidence indicates that betacyanins possess free-radical-scavenging and antioxidant activities, suggesting their beneficial effects on metabolic disorders. The main objective of this study was to isolate and identify the betaycanins from Hylocereus undatus (white-fleshed pitaya) peel and evaluate their ability to ameliorate obesity, insulin resistance, and hepatic steatosis in high-fat-diet (HFD)-induced obese mice. The purified pitaya peel betacyanins (PPBNs) were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS), and the male C57BL/6 mice were fed a low-fat diet, HFD, or HFD supplemented with PPBNs for 14 weeks. Our results showed that the white-fleshed pitaya peel contains 14 kinds of betacyanins and dietary PPBNs reduced HFD-induced body weight gain and ameliorated adipose tissue hypertrophy, hepatosteatosis, glucose intolerance, and insulin resistance. Moreover, the hepatic gene expression analysis indicated that PPBN supplementation increased the expression levels of lipid-metabolism-related genes (AdipoR2, Cpt1a, Cpt1b, Acox1, PPARγ, Insig1, and Insig2) and FGF21-related genes (β-Klotho and FGFR1/2) but decreased the expression level of Fads2, Fas, and FGF21, suggesting that the protective effect of PPBNs might be associated with the induced fatty acid oxidation, decreased fatty acid biosynthesis, and alleviated FGF21 resistance. KEYWORDS: Hylocereus undatus peel, betacyanins, obesity, insulin resistance, hepatic steatosis

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INTRODUCTION Obesity has been recognized as a major health problem worldwide. As a consequence of the imbalance between energy intake and expenditure, obesity is characterized by excessive fat accumulation and dysregulation of lipid metabolism.1 Moreover, obesity can lead to various complications, such as type 2 diabetes, cardiovascular disease, hypertension, and nonalcoholic fatty liver disease (NAFLD).2 Although a variety of obesity treatment strategies were developed, such as exercise, pharmacotherapy, and surgical interventions, many of the approaches have a number of limitations, such as ineffectiveness, severe side effects, and high rates of secondary failure.3,4 Recently, numerous studies demonstrated that natural bioactive compounds in food could act as important modulators in the prevention of obesity development.5 As water-soluble nitrogenous pigments, betalains have been extensively used as colorants in the food industry. Increasing evidence indicates that betalains possess free-radical-scavenging and antioxidant activities,6,7 which are closely associated with the protective effects of betalains against inflammation, high blood pressure, atherosclerosis, and hyperlipidemia.8,9 Betalains consist of two distinct classes, which are red−violet betacyanins and yellow−orange betaxanthins. Betanin (betanidin 5-O-β-Dglucoside) is the most abundant betacyanin, making up 75− 95% of total betalains.10,11 Pitaya, commonly known as dragon fruit, has received considerable attention not only because of its economic value but also its health benefits.12,13 Pitayas have been classified as red (Hylocereus polyrhizus), white (Hylocereus undatus), and yellow (Hylocereus megalanthus).14 Pitaya peel, which is usually © XXXX American Chemical Society

discarded during the processing and ends up as waste and a source of pollution, accounts for approximately 33% of the whole fruit weight.15 Although pitaya peel is a rich, natural, and cost-effective source of betalains, there are very limited studies focused on chemical composition and nutritional quality of pitaya peel. In this context, the objective of this study was to identify the varieties of betalains present in H. undatus peel by liquid chromatography−mass spectrometry analysis and evaluate the potential effects of pitaya peel betacyanins (PPBNs) on metabolic disorders.

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MATERIALS AND METHODS

Chemical Reagents and Plant Materials. Amberlite XAD7-HP resins and betanin were obtained from Sigma-Aldrich (Sigma-Aldrich, Steinheim, Germany). All of the other solvents and reagents were purchased from Aladdin (Aladdin, Shanghai, China) and were of analytical or high-performance liquid chromatography (HPLC) grade. White-fleshed pitayas were purchased from a fruit market in Hangzhou, and the peels were collected and stored at −80 °C prior to use. Extraction of Betacyanins from White-Fleshed Pitaya Peel. Pitaya peels were homogenized with 90% aqueous methanol and 0.1% trifluoroacetic acid (TFA), and the homogenate was kept in 4 °C for 12 h. Thereafter, the resulting extracts were centrifuged at 8000g for 40 min. Then, the supernatants were filtered through a 0.22 μm pore size Received: October 27, 2015 Revised: December 11, 2015 Accepted: December 12, 2015

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DOI: 10.1021/acs.jafc.5b05177 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry filter (Millipore, Bedford, MA) and concentrated using a vacuum rotary evaporator under reduced pressure at 38 °C. The concentrated extract was subsequently extracted 3 times with ethyl acetate. Then, the aqueous layer was collected and evaporated again. The concentrated solution was loaded onto a XAD-7HP column and eluted with distilled water first to remove organic acids, sugars, and other water-soluble compounds and then eluted with 90% methanol (0.1% TFA). The eluate was concentrated and reconstituted with distilled water, and the aqueous layer was lyophilized and stored at −80 °C until use. Identification of Betacyanins by HPLC−Electrospray Ionization/Tandem Mass Spectrometry (ESI/MS/MS) Analysis. HPLC−ESI/MS/MS (Agilent 1290 Infinity, Agilent Technologies, Santa Clara, CA) was used to identify the betacyanin compounds from the white-fleshed pitaya peel. Conditions for HPLC were as follows: The chromatography column was Acclaim 120 C18 (250 × 4.6 mm, 5 μm, 100 Å), and the mobile phase was solvent A (0.1% aqueous formic acid) and solvent B (acetonitrile and 0.1% formic acid). The gradient flow was programmed as follows: 0 min, 5% B; 3 min, 5% B; 22 min, 12% B; 42 min, 14% B; 55 min, 5% B; and 60 min, 5% B. The injection volume and flow rate were 0.5 mL/min and 10 μL, respectively. The betacyanin compounds were monitored using a wavelength of 538 nm. Mass spectra analyses were performed in positive mode. The settings were as follows: mass range m/z, 100−2000; ion trap temperature, 325 °C; capillary voltage, 3000 V; gas flow rate, 10 L/h; and desolvation temperature, 350 °C. Quantification of Betacyanins. The total betacyanin content was determined in deionized water using the previously described method, with slight modifications.16 The quantification of betacyanins was carried out by applying the equation BC = [(A × DF × MW × V/ εLW)], where BC is the betanin equivalents in mg/g, A is the absorption value at the absorption maximum (λmax = 538 nm), DF is the dilution factor, MW is the molecular weight of betanin (550 g/ mol), V is the dried powder solution volume (mL), ε is the molar extinction coefficient of betanin (60 000 L mol−1 cm−1 in H2O), L is the path length of the cuvette (1 cm), and W is the weight of the PPBN powder (g). Animals and Diets. All of the protocols in this research were approved by the Committee on the Ethics of Animal Experiments of Zhejiang University (permission number ZJU201550501), and the experimental procedures were conducted in accordance with the National Institutes of Health Regulations for the Care and Use of Animals in Research. A total of 60 male C57BL/6J mice (4 weeks old; n = 60) were supplied by the National Breeder Center of Rodents (Shanghai, China) and maintained, four animals per cage, in a temperature-controlled (23 ± 3 °C), 12 h light/dark cycle environment with free access to water and food. After 1 week for acclimation, the mice were randomly divided into the following five groups (n = 12): low-fat-diet (LFD) group, high-fat-diet (HFD) group, and HFD plus PPBNs of 50 mg/kg (HFD + L), 100 mg/kg (HFD + M), or 200 mg/kg (HFD + H) treatment groups. The compositions and energy densities of the purified diets were listed in Table S1 of the Supporting Information. In the PPBN group, mice received a low, moderate, or high dose of PPBNs by intragastric administration for 14 weeks. The mice in LFD and HFD groups received an equal volume of saline. The body weights and food intakes were monitored weekly. After 14 weeks of treatment, the mice were sacrificed by decapitation after a 12 h fast period. Blood samples were collected for serum preparation by centrifugation. The livers, hearts, kidneys, spleens, interscapular brown fat, and epididymal and perirenal fat pads were collected, weighted, and stored at −80 °C. Blood Chemistry and Hormone Analysis. The serum levels of glucose, triglyceride (TG), total cholesterol (TC), aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were determined by an automatic biochemistry analyzer (ACCUTE TBA-40FR, Toshiba Medical Systems Co., Tochigi, Japan) in accordance with the instructions of the manufacturer. The serum concentrations of insulin and neuropeptide Y (NPY) were determined using commercial assay kits (Elabscience

Biotechnology, Wuhan, Hubei, China), and the serum levels of adiponectin, leptin, and FGF-21 and were aslo measured by enzyme linked immunosorbent assay (ELISA) using commercial assay kits (R&D Systems, Minneapolis, MN) according to the protocols of the manufacturer. The serum levels of glucose and insulin were used to calculate the homeostasis model assessment-estimated insulin resistance (HOMA-IR) and homeostasis model assessment-estimated insulin sensitivity (HOMA-IS) according to the following formula:

HOMA‐IR = serum glucose (mmol/L) × serum insulin (mU) /22.5 HOMA‐IS = 1/[serum glucose (mmol/L) × serum insulin (mU)] Oral Glucose Tolerance Test (OGTT). At the end of the 12th week, the mice fasted for 12 h and the oral glucose tolerance test was performed after gavage with glucose (2 g/kg of body weight). Blood was collected from the tail vein at 0, 30, 60, 90, and 120 min for the determination of glucose concentrations. The area under the curve (AUC) of glucose was also calculated. Histological Analysis. Standardized pieces of epididymal and perirenal white adipose tissue, interscapular brown adipose tissue, and livers were fixed in 10% buffered formalin, and then the tissue sections were embedded in paraffin, sliced at 5 μm thickness, and stained with hematoxylin and eosin (H&E). For oil red O staining, the liver tissues were embedded in optimal cutting temperature gel, then the air-dried tissue sections of 5 μm were dipped in formalin and washed with oil red O solution. The images were captured using an Olympus CX41 camera (Olympus, Tokyo, Japan), and the hepatic steatosis scores were graded as follows: 0 (steatosis involving 66% fatty hepatocytes).17 Determination of Hepatic Lipids. Approximately 100 mg of liver tissues were homogenized, and the hepatic lipids were extracted with a chloroform/methanol (2:1, v/v) solution using the Folch method.18 Then, the levels of hepatic TG and TC in the extraction solution were determined by enzymatic methods using commercially available kits (Elabscience Biotechnology, Wuhan, Hubei, China) according to the instructions of the manufacturer. Quantitative Real-Time Polymerase Chain Reaction (PCR). Total RNA was extracted from the livers using Trizol (Invitrogen, Life Technologies, Carlsbad, CA) according to the instructions of the manufacturer, and the RNA concentrations were determined by NanoDrop 2000 (Thermo Scientific, Wilmington, DE). Then, 1 μg of total RNA was converted to the first strand of cDNA using a reverse transcription kit (TaKaRa, Dalian, Liaoning, China). Quantitative PCR was performed on the ABI 7500 system (Applied Biosystems, Foster, CA). The 20 μL reaction mixtures consisted of 1 μL of cDNA, paired primers (300 nM), and 10 μL of SYBR Green QPCR Master Mix (Roche Diagnostics, Ltd., Lewes, U.K.). The amplification program was set as follows: 3 min at 50 °C, 10 min at 95 °C, then 35 cycles of 15 s at 95 °C and then 60 s at 60 °C followed by melting curve for 60 s at 95 °C, then gradual decrease to 50 °C, 20 s at 50 °C, then gradual increase to 95 °C, and 20 s at 95 °C. The primer sequences are listed in Table S2 of the Supporting Information. The target genes were examined and normalized by β-actin, and the relative fold change was calculated using the 2−ΔΔCT method.19 Statistical Analysis. Values were expressed as the mean ± standard error of the mean (SEM). For experiments comparing multiple groups, the statistical significance were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test. The statistical analyses were performed using SPSS 19.0 statistical software. All of the results were considered statistically significant at p < 0.05. B

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Figure 1. Chromatographic profiles (HPLC gradient system) of purified betacyanins from white-fleshed pitaya peel at 538 nm. The peak assignments are in listed in Table S1 of the Supporting Information.

Table 1. Identification of Betacyanins in White-Fleshed Pitaya Peela peak

betacyanin

Rt (min)

λmax (nm)

m/z [M + H]+

20−22

betanidin-5-O-β-glucoside (betanin) 17-decarboxy-betanin isobetanidin-5-O-β-glucoside (isobetanin) 17-decarboxy-isobetanin betanidin-5-O-(6′-O-malonyl)-β-glucoside (phyllocactin) betanidin-5-O-(6′-O-3-hydroxy-butyryl)-β-glucoside 2-decarboxy-betanin 17-decarboxy-phyllocactin isobetanidin-5-O-(6′-O-malonyl)-β-glucoside (isophyllocactin) 2′-O-apiosyl-phyllocactin unknown 17-decarboxy-isophyllocactin 2-decarboxy-phyllocactin 2-decarboxy-isophyllocactin

16.33 18.28 19.32 21.96 23.23 24.21 24.61 25.22 26.01 26.66 27.11 28.65 34.7 37.94

538 505 538 505 538 536 533 505 536 536

551.15 507.16 551.15 507.16 637.16 637.16 507.16 593.16 637.15 769.19 656.17 593.16 593.16 593.16

1 222 320−22 422 522 620 722 821,22 921,22 1023 11 1222 1321,22 1422 a

505 533 533

m/z from MS/MS of [M + H]+ 389.10, 345.11, 389.10, 345.11 595.17, 389.10 345.11, 549.17, 595.17, 725.19, 612.18, 549.17, 549.17, 549.17,

343.09 299.10 343.09 389.10 299.10 507.16, 345.11 389.10 389.11, 683.19, 343.09, 150.06 568.19, 417.13, 389.10, 345.10 345.11 345.11 345.11

Rt = retention time. The peak number is in the order of elution (Rt) for the 14 betacyanins.

Figure 2. White-fleshed PPBNs reduce weight gain but not calorie intake in HFD-fed C57BL/6 mice. (A) Body weights of mice consuming the indicated diets for the 14 week intervention period. (B) Food intake on indicated diets of mice during the intervention period. (C) Calorie intake on indicated diets of mice during the intervention period. LFD, mice fed a low-fat diet; HFD, mice fed a high-fat diet; and HFD + L, M, and H, mice fed a high-fat diet supplemented with PPBNs at 50, 100, and 200 mg/kg, respectively. Data are expressed as the mean ± SEM (n = 12). Values not sharing a common letter differ significantly among groups (p < 0.05; ANOVA).

C

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Journal of Agricultural and Food Chemistry Table 2. Effect of White-Fleshed PPBNs on Tissue Weight and Serum Parameters in Micea item heart liver spleen kidney perirenal white adipose tissue epididymal white adipose tissue total white adipose tissue interscapular brown adipose tissue ALT (U/L) AST (U/L) TG (mmol/L) TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) adiponectin (μg/L) FGF21 (ng/L) NPY (ng/L)

LFD 0.57 3.95 0.22 1.23 0.54 1.44 1.98 0.42

± ± ± ± ± ± ± ±

0.02 d 0.06 c 0.01 b 0.05 c 0.07 a 0.14 a 0.2 a 0.01 d

40.17 153.83 0.77 3.19 2.41 0.29 93.03 713.83 402.19

± ± ± ± ± ± ± ± ±

6.57 a 11.73 a 0.05 a 0.28 a 0.26 a 0.03 a 3.36 a 36.04 a 34.01

HFD

HFD + L

Tissue Index 0.35 ± 0.01 a 2.82 ± 0.12 a 0.19 ± 0.01 a 0.77 ± 0.04 a 3.31 ± 0.11 c 5.98 ± 0.23 d 9.29 ± 0.27 d 0.29 ± 0.01 a Serum Parameters 98.58 ± 7.27 d 180.17 ± 11.1 b 1.59 ± 0.08 c 5.38 ± 0.23 d 4.41 ± 0.24 b 0.62 ± 0.04 c 70.65 ± 4.24 b 1141.74 ± 59.77 b 452.03 ± 30.63

HFD + M

HFD + H

0.38 3.25 0.18 0.9 2.64 4.98 7.62 0.35

± ± ± ± ± ± ± ±

0.02 0.08 0.01 0.04 0.09 0.15 0.18 0.01

ab b a b b c c b

0.4 3.4 0.2 0.91 3.77 4.08 7.85 0.37

± ± ± ± ± ± ± ±

0.01 0.09 0.01 0.03 0.09 0.13 0.17 0.01

81.42 145.58 1.29 4.64 4.19 0.54 80.97 854.78 466.49

± ± ± ± ± ± ± ± ±

5.46 c 6.79 a 0.07 b 0.15 c 0.33 b 0.03 c 6.09 ab 46.99 a 37.04

58.92 134.33 1.17 3.91 4.04 0.43 85.32 809.56 408.35

± ± ± ± ± ± ± ± ±

3.32 b 6.02 a 0.07 b 0.29 b 0.28 b 0.03 b 3.68 a 49.6 a 39.11

b b a b d b c bc

0.5 3.7 0.2 1.15 2.43 4.51 6.94 0.39

± ± ± ± ± ± ± ±

0.01 0.09 0.01 0.04 0.08 0.09 0.14 0.01

41.92 147.5 0.85 3.42 4.04 0.31 92.87 785.21 415.08

± ± ± ± ± ± ± ± ±

2.48 a 7.94 a 0.07 a 0.17 ab 0.19 b 0.04 a 3.49 a 51.74 a 32.46

c c ab c b b b cd

a

LFD, mice fed a low-fat diet; HFD, mice fed a high-fat diet; and HFD + L, M, and H, mice a fed high-fat diet supplemented with PPBNs at 50, 100, or 200 mg/kg. Data are expressed as the mean ± SEM (n = 12). Means not sharing a common letter differ significantly among groups (p < 0.05; ANOVA).

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RESULTS Quantification and Identification of Purified WhiteFleshed PPBNs. The content of betacyanins expressed as betanin equivalents per 1 g of PPBN powder was 25.32 ± 0.23 mg/g. The betacyanin identification and peak assignment were performed on the basis of the comparison of their retention time, ultraviolet−visible (UV−vis) spectra, and mass spectral analysis to those of standards and literature data.20−23 The absorbance peaks from HPLC were recorded at 538 nm (Figure 1), and the data of the peak identification, retention times, and compound identification obtained from the LC/ MS/MS analyses were summarized in Table 1. Effect of PPBNs on Body Weight and Food Intake. At the end of the experiment, the mice fed a HFD gained more weight and body fat percentage (per 100 g of body weight) compared to those fed a LFD and PPBN administration significantly reduced the HFD-induced increase of body weight gain and body fat (Figure 2A and Table 2). Moreover, HFD feeding decreased the relative weight of the heart, liver, spleen, kidney, and interscapular brown, while PPBN administration reversed the trend (Table 2). The mice fed a HFD consumed more calories than those fed a LFD, but the calorie intake was not significantly different among the HFD-fed mice with or without supplementation of betacyanins, suggesting that PPBNs could significantly decrease the HFD-induced body weight gain without affecting the calorie intake (Figure 2). Effect of PPBNs on Serum Biochemical Parameters. The serum levels of TG, TC, HDL-C, LDL-C, ALT, and AST were significantly increased in mice fed a HFD compared to those fed a LFD, and administration of PPBNs significantly reduced the levels of TG, TC, LDL-C, ALT, and AST. No influence of PPBNs on HDL-C was observed (Table 2). Serum Levels of Leptin, Adiponectin, NPY, and FGF21. HFD feeding induced a decrease in the serum level of adiponectin but increased the concentration of FGF21, while supplementation with PPBNs significantly reversed the trend.

No differences were observed in the serum level of leptin and NPY among all of the dietary groups (Table 2). Impact of PPBNs on HFD-Induced Hepatic Steatosis and Adipose Tissue Hypertrophy. Histological analysis revealed an accumulation of lipid droplets in the livers of mice fed a HFD, and the mice developed a high degree of hepatic steatosis with severe cytoplasmic vacuoles and swelling of hepatocytes, whereas no significant abnormalities were observed in LFD-fed mice (Figure 3A). PPBN administration attenuated hepatic lipid accumulation, alleviated the formation of steatosis, and decreased the hepatic steatosis grade scores in a dose-dependent manner (panels A and B of Figure 3). Consistent with these results, PPBN supplementation also significantly decreased the hepatic concentrations of TG and TC (panels C and D of Figure 3). In addition, H&E staining of adipose showed that PPBNs significantly reduced the size of white and brown adipocytes (Figure 3A), suggesting that PPBN supplementation attenuated HFD-induced adipose hypertrophy. Effect of PPBNs on Glucose Tolerance and Insulin Resistance. As the OGTT shows, the glucose level in the mice fed a HFD was significantly higher at 30, 60, 90, and 120 min after oral gavage of glucose than that in the LFD-fed mice and the increase fasting blood glucose concentration was significantly reduced by the administration of PPBNs compared to the HFD group (panels A and B of Figure 4). In addition, HFD feeding induced a significant increase in the fasting serum levels of glucose and insulin, and the mice in the HFD + L, M, and H groups revealed a significant decrease in the fasting serum glucose and insulin levels (panels C and D of Figure 4). These data suggested that PPBN administration alleviated HFDinduced glucose intolerance and insulin resistance in mice. This conclusion was also supported by the PPBN-induced lower HOMA-IR but higher HOMA-IS index (panels E and F of Figure 4). Influence of PPBNs on Hepatic Gene Expression. HFD feeding decreased the expression levels of lipid metabolismD

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Figure 3. White-fleshed PPBNs attenuate hepatic steatosis and adipocyte hypertrophy. (A) Histological analysis for epididymal white adipose tissue (eWAT; H&E), perirenal white adipose tissue (pWAT; H&E), interscapular brown adipose tissue (iBAT; H&E), and liver (H&E and oil red O staining). (B) Steatosis grade score (n = 6). The hepatic steatosis scores were graded as follows: 0 (steatosis involving 66% fatty hepatocytes). (C) Hepatic TG levels (n = 12). (D) Hepatic TC levels (n = 12). LFD, mice fed a low-fat diet; HFD, mice fed a high-fat diet; HFD + L, M, and H, mice fed a high-fat diet supplemented with PPBNs at 50, 100, and 200 mg/kg, respectively; and HE, hematoxylin−eosin. Data are expressed as the mean ± SEM. Values not sharing a common letter differ significantly among groups (p < 0.05; ANOVA).

related genes (AdipoR2 and PPARγ) and the cholesterol biosynthesis-related genes (Insig1 and Insig2), and PPBN treatment significantly enhanced the expression of these genes in a dose-dependent manner (panels C and D of Figure 5). In addition, a HFD increased the expression level of fibroblast growth factor 21 (FGF21) and suppressed the expression of Klb, FGFR1, and FGFR2, while PPBN administration reversed the trend (Figure 5E).

significantly reduced diet-induced body weight gain and attenuated obesity-related insulin resistance and hepatic steatosis. H. polyrhizus fruit and its methanol extract have previously been shown to lower the plasma TG, TC, and LDL-C levels and increase the HDL-C level in HFD-fed rats and type 2 diabetic subjects.25,26 However, despite the reducing effect of pitaya fruit on the lipid profile, no changes in the body weights and fat mass were observed in these studies. We postulated that this condition may be due to the high content of sugar and fat in pitaya fruit, which might counteract the weight loss effect. Consistent with these studies, our results showed that PPBN supplementation significantly decreased serum levels of TG, TC, and LDL-C in HFD-fed mice. Therefore, in conjunction with the previous studies, these observations suggested that PPBNs might be the main bioactive compounds in pitaya that exhibited the beneficial effects on the lipid profile. Moreover, unlike the previous studies, we found that the body weight gain started to decrease after 1 week of supplementation of PPBNs

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DISCUSSION The antiobesity effect of natural pigments from vegetables and fruits has been demonstrated but betacyanins receive little attention. Red- and white-fleshed pitaya are rich in betacyanins, while the consensus on the betacyanin varieties has not been reached.14,22,24 This study was undertaken to investigate the beneficial effects of PPBNs on obesity and obesity-related metabolic syndromes in HFD-fed C57BL/6J mice, which are susceptible to diet-induced obesity, type 2 diabetes, and NAFLD. Our results clearly showed that PPBN administration E

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Figure 4. White-fleshed PPBN supplement alleviates diet-induced hyperglycemia, improves insulin resistance, and increases insulin sensitivity. (A) Effect of PPBNs on glucose tolerance in mice fasted for 12 h as determined by the OGTT. (B) AUC for the OGTT test. Effect of PPBNs on the fasting serum (C) glucose and (D) insulin in mice. (E) HOMA-IR index. (F) HOMA-IS index. LFD, mice fed a low-fat diet; HFD, mice fed a highfat diet; and HFD + L, M, and H, mice fed a high-fat diet supplemented with PPBNs at 50, 100, and 200 mg/kg, respectively. n = 12 (A), n = 10 (B, E, and F), and n = 8 (C). Data are expressed as the mean ± SEM. Values not sharing a common letter differ significantly among groups (p < 0.05; ANOVA).

Figure 5. White fleshed PPBNs alter the mRNA levels in the liver of mice. (A) Relative expression levels of Fads2 and Fas. (B) Relative expression levels of Cpt1a, Cpt1b, and Acox1. (C) Relative expression levels of lipid metabolism genes (AdipoR2 and PPARγ). (D) Relative expression levels of cholesterol biosynthesis-related genes (Insig1 and Insig2). (E) Relative expression levels of fibroblast growth factor 21 (FGF21), β-Klotho (Klb), and receptors of FGF21 (FGFR1 and FGFR2). LFD, mice a fed low-fat diet; HFD, mice fed a high-fat diet; and HFD + L, M, and H, mice fed a high-fat diet supplemented with PPBNs at 50, 100, and 200 mg/kg, respectively. Data are expressed as the mean ± SEM. Statistical analyses were performed with Student’s t test. (∗) p < 0.05 versus the LFD group. (#) p < 0.05 versus the HFD group.

and finally led to a significant decrease in the body weight of mice in a dose-dependent manner. Furthermore, histological analysis showed that PPBN treatment significantly reduced the size of adipocyte in the white and brown adipose tissues. In addition, there was no difference in the daily calorie intake or

the serum level of NPY, which acts as a neurotransmitter in the brain and regulates food intake and storage of energy as fat. On the basis of these evidence, we inferred that PPBNs suppressed HFD-induced body weight gain without affecting the energy intake of mice. Histological analysis of liver demonstrated that F

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Journal of Agricultural and Food Chemistry

FGF21 activity depends upon its binding to a receptor complex consisting of a cofactor called β-Klotho (Klb) and FGFRs and then elicits the intracellular signaling cascades.41,42 To identify the potential mechanism by which obesity induced FGF21 resistance, the hepatic gene expressions of Klb and FGFR1/2 were evaluated. Our results indicated that HFD feeding resulted in a significant suppression of Klb and FGFR1/ 2, while PPBN supplement significantly increased the expression levels of these genes. These data implied that the impaired action of FGF21 might be due to the decreased expression of its receptors and PPBNs may directly or indirectly improve the expression profile of these receptors, thus attenuating the FGF21 resistance and contributing to its antiobesity, antidiabetic, and antihepatosteatosis effects. Although the exact and detailed mechanisms of absorption, metabolism, and excretion of betacyanins have not yet been fully elucidated, there is increasing experimental evidence proving their numerous biological activities that implied the potential health benefits of betacyanins.43,44 A total of 14 betacyanins were identified in the white-fleshed pitaya peel by LC/MS/MS analysis, but we were not clear if all of them were the bioactive agents. Therefore, further studies are required to identify the distinct effective compounds in PPBNs. In summary, the present study demonstrated that PPBN treatment protected from diet-induced obesity, hepatic steatosis, and insulin resistance in mice and the protective effects of PPBNs were associated with the induced fatty acid oxidation, decreased fatty acid biosynthesis, and improved FGF21 sensitivity. Our results suggested a potential dietary choice of PPBNs in the management of obesity, type 2 diabetes, and NAFLD.

administration of PPBNs effectively attenuated hepatic lipid accumulation in obese mice. In combination with the results that PPBN supplementation decreased the hepatic steatosis grades and reduced hepatic TG and TC and serum ALT and AST levels, we believed that PPBNs could significantly alleviated HFD-induced hepatic steatosis. Furthermore, PPBN treatment increased the hepatic expression levels of AdipoR2, Cpt1a, Cpt1b, and Acox1 as well as elevated the serum levels of adiponectin but decreased the expression levels of Fas and Fads2 compared to the HFD mice. Adiponectin, secreted by adipocytes, has been proven to regulate the metabolism of lipids and glucose.27,28 AdipoR2 serves as a receptor for adiponectin and mediates increased AMPK and PPAR-α ligand activities as well as fatty acid oxidation and glucose uptake by adiponectin.29 Cpt1a, Cpt1b, and Acox1 encode the rate-controlling enzymes of the fatty acid β-oxidation pathway,30−32 while Fas and Fads2 are proven to be involved in the synthesis of fatty acids. Moreover, PPARγ, the upstream regulator of Fas and Acox1,33 was also downregulated by PPBN treatment. Our results implied that the reduced biosynthesis but enhanced fatty acid oxidation might contribute to the beneficial effects of PPBN. In our study, we also observed higher serum and hepatic TC levels accompanied by an increased expression of Insig1 and Insig2 in HFD mice, while PPBN treatment reversed the trend. In consideration of the previous findings that high levels of insulin induced overexpression of Insig1 and Insig2 in the liver, leading to cholesterol synthesis,34 we postulated that PPBN treatment could lower insulin levels and, consequently, decrease expression of Insig1, Insig2, and cholesterol levels. Obesity is often accompanied by insulin resistance, thus increasing the risk of type 2 diabetes; therefore, the bioactive compounds with both antiobesity and antidiabetic effects are particularly beneficial for health. Previous studies demonstrated that red pitaya consumption significantly decreased the blood glucose level in type 2 diabetic subjects25 and significantly improved hyperinsulinemia in insulin-resistant rats.35 Consistently, our results indicated that PPBN administration significantly decreased the fasting serum levels of glucose and insulin. The beneficial effects of PPBNs on hyperglycemia and hyperinsulinemia were also confirmed by the OGTT and lowered HOMA-IR index. Furthermore, the insulin-sensitizing effect was supported by the higher HOMA-IS index of the PPBN-treated mice compared to that fed a HFD alone. To confirm the potential action sites for PPBNs to exert its antidiabetic effect, we examined the expression profile of FGF21. FGF21, mainly expressed in liver, is involved in the regulation of glucose, lipid, and energy metabolism. Administration of exogenous FGF21 improves metabolic disorders, for instance, increasing glucose tolerance and insulin sensitivity, regulating lipid oxidation, improving lipid profile, attenuating hepatic steatosis, and reducing body weights.36,37 However, consistent studies indicate that circulating FGF21 levels are markedly increased in obesity and impaired glucose tolerance, insulin resistance, and hypertriglyceridemia and liver injury states.38−40 Similar to the insulin resistance, the fail of endogenous FGF21 to suppress obesity, type 2 diabetes, and hepatosteatosis implies a state of FGF21 resistance as a result of the impaired FGF21 action. As expected, the fasting serum FGF21 level was significantly increased in the HFD-induced obese mice, which was consistent with the previous findings and confirmed the speculation of compensatory FGF21 overproduction in the obese state.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05177. Composition of purified diets (Table S1) and primer sequences used for quantitative real-time PCR (Table S2) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-571-86098861. Fax: +86-571-86971139. Email: [email protected]. Funding

This work was supported by the National Key Technology R&D Program of China (Grant 2012BAD33B08), the Zhejiang Provincial Natural Science Foundation of China (Grant Z14C200006), and the Foundation of Fuli Institute of Food Science, Zhejiang University (Grant KY201301). Notes

The authors declare no competing financial interest.

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