c4fo01092b 1..11

Food &Function

PAPER

Cite this: DOI: 10.1039/c4fo01092b

Received 28th November 2014,Accepted 15th December 2014

DOI: 10.1039/c4fo01092b

www.rsc.org/foodfunction

The e!ect of isorhamnetin glycosides extractedfrom Opuntia !cus-indica in a mouse model ofdiet induced obesity†

César Rodríguez-Rodríguez,a Nimbe Torres,b Janet A. Gutiérrez-Uribe,a

Lilia G. Noriega,b Iván Torre-Villalvazo,b Ana M. Leal-Díaz,a

Marilena Antunes-Ricardo,a Claudia Márquez-Mota,b Guillermo Ordaz,b

Rocío A. Chavez-Santoscoy,a Sergio O. Serna-Saldivara and Armando R. Tovar*b

A diet rich in polyphenols can ameliorate some metabolic alterations associated with obesity and type 2

diabetes. Opuntia !cus-indica (OFI) is a plant rich in isorhamnetin glycosides and is highly consumed in

Mexico. The purpose of this research was to determine the metabolic e!ect of an OFI extract on a mouse

model of diet-induced obesity and in isolated pancreatic islets. OFI extract was added to a high fat (HF)

diet at a low (0.3%) or high (0.6%) dose and administered to C57BL/6 mice for 12 weeks. Mice fed the HF

diet supplemented with the OFI extract gained less body weight and exhibited signi"cantly lower circulat-

ing total cholesterol, LDL cholesterol and HDL cholesterol compared to those fed the HF diet alone. The

HF-OFI diet fed mice presented lower glucose and insulin concentration than the HF diet fed mice.

However, the HF-OFI diet fed mice tended to have higher insulin concentration than control mice. The

OFI extract stimulated insulin secretion in vitro, associated with increased glucose transporter 2 (GLUT2)

and peroxisome proliferator-activated receptor gamma (PPAR!) mRNA content. Furthermore, the OFI

extract improved glucose tolerance, and additionally increased energy expenditure. These metabolic

improvements were associated with reduced adipocyte size, increased hepatic IRS1 tyr-608 and S6 K thr-

389 phosphorylation. OFI isorhamnetin glycosides also diminished the hepatic lipid content associated

with reduced mRNA expression of the endoplasmic reticulum stress markers and lipogenic enzymes and

increased mRNA expression of genes related to fatty acid oxidation. Overall, the OFI extract prevented the

development of metabolic abnormalities associated with diet-induced obesity.

1. IntroductionObesity is one of the most important risk factors for type 2 dia-betes. Type 2 diabetes is a complex metabolic disorder associ-ated with the development of insulin resistance, as a result ofan impaired insulin signaling, and "-cell dysfunction, thatleads to abnormal glucose and lipid metabolism.1 Particularly,it has been established that a high fat diet induces hepaticendoplasmic reticulum (ER) stress along with increased lipo-genesis, and it has been proposed that this mechanism is theprimary cause of hepatic insulin resistance and progression offatty liver to steatohepatitis and fibrosis.2,3 Furthermore, vis-

ceral obesity is associated with a decrease in insulin-mediatedglucose uptake and is clearly related to insulin resistance. Freefatty acids, which are released from visceral fat, impair phos-phoinositide 3-kinase (PI3K), a key element in the insulin sig-naling pathway.4 Additionally, elevated plasma free fatty acidsupregulate glucose production, insulin secretion and lead to"-cell dysfunction.5 When there is a rise in plasma glucose,"-cells internalize glucose through GLUT2, then, glucose oxi-dation induces the activation of a voltage-dependent calciumchannel triggering insulin secretion.6 However, long termexposure to fatty acids diminishes "-cell responsiveness toglucose leading to type 2 diabetes. Therefore, diminishingplasma free fatty acids by increasing hepatic fatty acid oxi-dation and inhibiting hepatic fatty acid synthesis (lipogenesis)will consequently diminish insulin resistance and type 2diabetes.

Diet can be modified to ameliorate the e!ects of type 2 dia-betes. Polyphenols, and particularly flavonoids, present in thediet lower the predisposing factors for the risk of developingtype 2 diabetes.1 For instance, extracts rich in polyphenols

†Electronic supplementary information (ESI) available. See DOI: 10.1039/C4FO01092B

aCentro de Biotecnología-FEMSA, Tecnológico de Monterrey, Av. Eugenio Garza Sada2501 Sur, C.P. 64849 Monterrey, NL, MéxicobDepartamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicasy Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, C.P. 14000 México, DF,México. E-mail: [email protected]; Fax: +52 55 5655 3038; Tel: +52 55 5655 3038

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from Oiltea camellia reduce triglycerides in 3T3-L1 adipocytes.7

In addition, polyphenols extracted from plants like mulberryleaf and Cinnamomum cassia have been demonstrated toreduce hyperglycaemia.8,9 It has been shown that quercetin, apolyphenol present in Opuntia ficus-indica (OFI), increasesinsulin secretion in isolated pancreatic islets by the direct acti-vation of L-type calcium channels10 but when mice were fedwith diets enriched with quercetin a reduction in plasmainsulin was observed.11

The genera Opuntia includes di!erent plants well adaptedto arid and semi-arid zones. In 2005, Opuntia plantationsranked third in the total volume of production of vegetables inMéxico with a volume of 759 072 tons and eighth in the valueof production.12 In healthy humans, OFI cladode consumptionincreased blood and plasma antioxidant activity.13 It has beendescribed that OFI can ameliorate some abnormalities ofglucose homeostasis, particularly in type 2 diabetic sub-jects.14,15 OFI powder mixed with other plants had a hypo-glycemic e!ect in the db/db mice, potentially due to improvedinsulin secretion from pancreatic "-cells.16

It has been suggested that C57BL/6 mice carry a genetic pre-disposition to develop type 2 diabetes. This can be triggered bydiet-induced obesity.17 Interestingly, C57BL/6 obese micetreated with extracts (rich in polyphenols) from plants like Vac-cinium ashei, Glycyrrhiza uralensis, and Co!ea arabica had adecrease in inflammation,18 abdominal fat,19 serum choles-terol and glucose concentration.20 Furthermore, treatmentwith a cranberry extract rich in quercetin ameliorated insulinresistance and plasma lipid profile in C57BL/6 obese mice.This e!ect has been associated with an increase in circulatingadiponectin that, via its receptor AdipoR2 in skeletal muscles,can stimulate the phosphorylation state of adenosine mono-phosphate-activated protein kinase (AMPK) leading to anincrease in fatty acid oxidation.21

Isorhamnetin, a methylated flavonol, is one of the mostabundant flavonoids in Opuntia ficus-indica (OFI) and it isfound in the form of at least five di!erent di- and tri-glycosides.22–26 Isorhamnetin has been demonstrated to inhibitadipogenesis in vitro27 and diminish oxidative stress in rats withstreptozotocin-induced diabetes.28 However, the e!ect of iso-rharmnetin glycosides in diet-induced obesity is not known.

The purpose of this work was to study the e!ect of an OFIextract rich in isorhamnetin glycosides on body weight gain,biochemical parameters and metabolic rate in a mouse modelof diet induced obesity. These e!ects were analyzed along withthe mRNA expression of lipogenic genes and genes related tofatty acid oxidation. In particular, isorhamnetin glycosideswere used to evaluate the increase in insulin secretion inpancreatic islets.

2. Materials and methods2.1. Extract preparation

Extracts were obtained from the OFI flour donated by Alimen-tos Funcionales S. de R.L. de C.V. The dehydrated cactus flour

was prepared from 7 month old pads of Opuntia ficus-indicaVar. Jalpa, collected in March, 2011 in Rancho Santa Emilia,located at Montemorelos, N.L., México (25° 11! 13.99" N,99° 49! 36.01" W) at an altitude of 480 m above sea level. TheSchool of Agronomy of Universidad Autónoma de Nuevo León,México, performed the taxonomic identification of the cactusspecies.

The extraction method was similar to the one previouslyreported26 with slight modifications. Briefly, the OFI flour wassubject to extraction with methanol 80% (1 : 10), stirring at200 rpm for 12 hours followed by 3 additional hours on thecounter. Then the supernatant was filtered through WhatmanNo. 1, using a vacuum pump. Next, it was concentrated(40 minutes, 60 °C, 180 mbar) with the aid of an evaporator(Büchi, Flawil, Switzerland). The residual was extracted twicewith 80% methanol and the supernatants were recovered andconcentrated as described above. In order to increase and stan-dardize the content of isorhamnetin glycosides in the extract,aqueous concentrates were pooled and purified with solidphase extraction (SPE) using a C18 column (Strata C18-E;55 #m, 70A). Isorhamnetin glycosides retained in the SPE car-tridges were recovered with 100% methanol. This process wasrepeated with the OFI flour produced in February 2013. TheOFI isorhamnetin glycoside extract was evaporated to drynessand lyophilized until use.

2.1.1. Isorhamnetin glycoside quantification. The lyophi-lized isorhamnetin glycoside extract was resuspended using amix of methanol–water (1 : 5). After 2 #L injection, separationwas carried out employing a Zorbax Eclipse XDB C18 3.0 !100 mm, 3.5 #m column (Agilent Technologies Santa Clara,CA) with a flow of 0.45 mL min!1. The mobile phase consistedof (A) HPLC-grade water with 0.1% of formic acid and (B)HPLC-grade methanol. Separation was achieved starting with35% of B for the first 5 min, increasing the B concentration to60%, until the 20th minute, and then increasing the B concen-tration to 90% for the following 5 min. Chromatograms wereobtained at 365 nm. Standard curves for isorhamnetin (Iso)(>95%; HPLC grade) were prepared (range from 3.2 up to320 #M) (Sigma, St. Louis, MO). Glycosides were quantified asa percentage of abundance in the obtained extract.

2.1.2. Isorhamnetin glycoside purification. Semi-prepara-tive purification of the isorhamnetin glycoside from theOpuntia ficus-indica cladode extract was performed using anHPLC-PDA equipped with a photo diode array detector(Agilent 1100 Series G1311A Quat Pump. Santa Clara, CA).Chromatograms were obtained at 365 nm after injection of50 #L of the sample and data generated by the Agilent Chem-Station software. The separation was performed in a semi-preparative Zorbax SB-C18 (9.4 ! 250 mm, 5 #m) columnoperating at 17 °C with a flow rate of 2.0 mL min!1. Themobile phase used was (A) water with 0.1% formic acid(Sigma, St. Louis, MO) and (B) 80% methanol. The separationwas achieved starting with 35% of B, increasing to 60% until2 min, increasing to 90% until 19 min, and then decreasing to0% of B for the next 3 min. The most abundant flavonoidswere collected based on peaks and concentrated under

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nitrogen. The purity and concentration of injection pools wereobtained using the method described in 2.1.1. Isorhamnetinglycoside used for further experiments had a purity higherthan 90%.

2.2. In vitro study

2.2.1. Islet isolation. Islets were isolated from male Wistarrats as described before29 with some modifications. MaleWistar rats were obtained from Harlan-Teklad (México, D.F.),with a weight between 200 and 230 g (8 weeks of age). Animalswere housed individually and maintained on a constant 12 hlight/dark cycle at 22 °C and had free access to food and water.The animal protocol was approved by the Animal Committeeof the National Institute of Medical Sciences and Nutrition,México City. Briefly, the pancreas were infused through thebile duct with Hank’s balanced salt solution (H6136-1L SigmaSt. Louis, MO) and type V collagenase (1 mg mL!1; fromClostridium histolyticum, Sigma St. Louis, MO). Then, the pan-creatic tissue was digested with 10 mL of Hank’s balanced saltsolution and type V collagenase (2 mg mL!1) for 1 h at 37 °Cand 120 rpm. The islets were washed with Hank’s balancedsalt solution, centrifuged at 900 rpm for 20 s at 4 °C and thenthe supernatant was decanted and additional Hank’s balancedsolution was added to repeat this step once. The islets wereseparated with a ficoll (Sigma St. Louis, MO) density gradientand handpicked under a stereomicroscope to exclude any con-taminating tissue.

2.2.2. Pancreatic islets cultured with isorhamnetin glyco-side extract. The isolated islets were maintained overnight in asuspension culture in 6-well plates at 37 °C under an atmos-phere of 5% CO2 and 95% air. The culture medium consistedof RPMI 1640 (Invitrogen, Carlsbad, CA), supplemented with5.5 mM glucose, 10% of heat-inactivated fetal bovine serumand 5% of Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA).To evaluate insulin secretion, 10 islets were washed and incu-bated for 30 minutes in Krebs balanced salt solution (NaCl130 mM, KCl 3.4 mM, NaH2PO4 0.5 mM, MgSO4 1.0 mM,HEPES 10 mM, Na2CO3 5 mM, CaCl2 1.5 mM). The islets wereincubated with 5.5 mM glucose alone (control) or with OFIextract, purified isorhamnetin-glucosyl-rhamnosyl-rhamnoside(IG1), or isorhamnetin (Sigma, St. Louis, MO). The di!erentcompounds were tested at a concentration of 2 #M of isorham-netin equivalents. The medium was collected and frozen inorder to determine insulin concentration using ELISA kits(ALPCO, Salem, NH). This experiment was performed in tripli-cate. To evaluate mRNA expression, 50 islets were washed andincubated for 2 h in Krebs balanced salt solution as describedabove. After incubation, the islets were collected and RNA wasextracted.

2.2.3. Isolation of total RNA from the islets and real timePCR. Total RNA was extracted from the islets using TRIzolaccording to the manufacturer’s protocol (Invitrogen, Carls-bad, CA). For quantitative real-time PCR, the first strand cDNAwas synthesized from 1300 ng of total RNA using the oligo (dT)primer and the MMLV reverse transcriptase (Invitrogen, Carls-bad, CA). The samples were subjected to quantitative amplifi-

cation using rat primer sets for GLUT2 and PPAR!. PCR wascarried out in triplicate for each sample and performed in atotal volume of 10 #l containing 120 ng of cDNA, 900 nM ofeach primer and 5 #l of Sybr Green Universal PCR Master Mix(Invitrogen, Carlsbad, CA). Gene expression was normalizedwith the expression of the housekeeping genes "-actin andhypoxanthine guanine phosphoribosyl transferase (HPRT).Relative expression levels were calculated using the 2!$$Ct

method.30

2.3. In vivo study

2.3.1. Animals. Twenty male C57BL/6NCrl (8 weeks of age)mice were obtained from the Experimental Research Depart-ment from the Animal Care Facilities at the National Instituteof Medical Sciences and Nutrition, México, D.F. The mice wererandomly assigned to four di!erent treatments and were main-tained on the same dietary regimen for the next 12 weeks.Each cage housed five animals and was maintained on a con-stant 12 h light/dark cycle at 22 °C and had free access to foodand water. The animal protocol was approved by the AnimalCommittee of the National Institute of Medical Sciences andNutrition, México City.

2.3.2. Diet. The control (12% energy from fat) and high fat(HF) diets (45% energy from fat) were based on the Test Diet®formulation specially designed to induce obesity (Table 1). TheOpuntia ficus-indica isorhamnetin glycosides were evaluated attwo doses, 0.3% and 0.6% of the high fat diet. These dietswere designated as high fat + low dose (HF + LD) and high fat+ high dose (HF + HD).

Table 1 Composition of diets for di!erent treatments: control, high fat,high fat + low dose (HF + LD) and high fat + high dose (HF + HD)

Ingredient (g kg!1) Control High Fat HF + LD HF + HD

Caseina 198.7 241.4 241.4 241.4Maltodextrinb 150.0 294.6 294.6 294.6Soy oilc 51.6 57.5 57.5 57.5Cellulosed 28.4 17.2 14.2 11.2Cholinee 1.9 2.3 2.3 2.3Sacarose f 175.9 103.5 103.5 103.5Corn starchg 325.9 — — —Vitaminsh 18.9 29.9 29.9 29.9Mineralsi 47.3 57.5 57.5 57.5Methionine j 1.4 1.7 1.7 1.7Inulink — 17.2 17.2 17.2Lard — 177.2 177.2 177.2OFI extract — — 3.0 6.0Total 1000 1000 1000 1000

% Energy (kcal) from:Protein 20.6 20.6 20.6 20.6Fat 12.0 45.2 45.2 45.2Carbohydrates 67.4 34.2 34.2 34.2

a Casein vitamin-free test (Harlan Teklad). bMaltodextrin (CPIngredients). c Soy Oil (Nutrioli). dCellulose (AIN Alphacel Non-Nutritive Bulk, MP Biochemicals). eCholine (Sigma-Aldrich, St. Louis,MO). f Sacarose (brown sugar). g Corn starch (CP Ingredients).h Vitamins (AIN-76 Vitamin Mix, MP Biochemicals). iMinerals (AIN-76Minerals Mix, MP Biochemicals). jMethionine (Sigma-Aldrich,St. Louis, MO). k Inulin (CP Ingredients).

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2.3.3. Glucose tolerance test. This test was performed bythe intraperitoneal injection of a glucose load of 2 g per kgbody weight in 7 h fasted mice, at the 10th week of treatment.Blood samples were collected from the tail vein at 0, 15, 30,45, 60, 90 and 120 min after the administration of theglucose load.21 Plasma glucose concentration was measuredusing a OneTouch Ultra Glucose Meter (LifeScan, Inc.Milpitas, CA).

2.3.4. Indirect calorimetry. Whole-body metabolic stateswere tested by indirect calorimetry in a CLAMS System (Colum-bus Instruments, Columbus, OH) for 12 hours after 12 hoursof habituation. This study was performed at the 11th week oftreatment. The mice were placed in individual acrylic cagesconnected to the Oxymax Lab Animal Monitoring Systemwhere O2 and CO2 were measured during fasting and feedingperiods.31 The respiratory exchange ratio (RER) was calculatedas the volume of CO2 exhaled (VCO2 mL kg!1 h!1) divided bythe volume of O2 inhaled (VO2 mL kg!1 h!1).

2.3.5. Biochemical parameters. Plasma triglycerides, totalcholesterol, low density lipoprotein (LDL) cholesterol, highdensity lipoprotein (HDL) cholesterol and glucose were ana-lyzed with Cobas 111 (Roche, Basilea, Switzerland). Plasmainsulin, glucagon and leptin were measured using ELISA kits(ALPCO, Salem, NH). Insulin resistance was estimatedindirectly through HOMA-IR, calculated as follows: (fastingglucose (mmol L!1)) ! (fasting insulin (µU mL!1))/22.5.32

2.3.6. Hepatic lipid quantitation. Total lipids wereextracted from the liver according to the Folch extraction proto-col.33 Briefly, snap-frozen liver tissues ("100 mg) were homo-genized and extracted twice with a chloroform–methanol (v/v =2 : 1) solution. The organic layer was dried under nitrogengas and solubilized in isopropanol/Triton X-100 (10%) andthen assayed for total cholesterol and triglyceride concen-tration using enzymatic kits (DiaSys Diagnostic SystemsGmbH, Holzheim, Germany).

2.3.7. Isolation of total RNA from the liver and real timePCR. Samples of total RNA were reverse transcribed and thensubjected to quantitative amplification as described aboveusing mice primer sets for sterol regulatory element-bindingprotein 1 (SREBP1), fatty acid synthase (FAS), stearoyl CoAdesaturase (SCD1), peroxisome proliferator-activated receptoralpha (PPAR%), carnitine palmitoyl transferase 1 (CPT1), acylCoA oxidase (AOX), insulin receptor substrate (IRS1), proteinkinase B (AKT), GLUT2, X-box-binding protein 1 (XBP1),C/EBP-homologous protein (CHOP) and activating transcrip-tion factor 6 (ATF6). Gene expression was normalized with theexpression of the housekeeping genes cyclophilin and HPRT.

2.3.8. Western blot for S6 K, IRS1, p-S6 K (Thr389) andp-IRS1 (Tyr941). Total protein was extracted from the liver andquantified by the Lowry method, using an albumin standardcurve. Proteins were separated in the SDS-PAGE and thentransferred to polyvinylidene difluoride membranes (PVDF)(GE Health Care Life Sciences, Piscataway, NJ) using a wet elec-troblotting system (Bio-Rad, Hercules, CA). The PVDF mem-branes were blocked with skimmed milk (5%) and immuno-blotting was performed using monoclonal antibodies against

S6 Kinase, phospho-p70 S6 Kinase (Thr389), IRS1 andphospho-IRS1 (Tyr941) (Millipore, Temecula, CA). Immuno-reactive bands were visualized using the horseradish peroxi-dase labeled goat antimouse antibody (1 : 3500) after theoxidation of luminal as a luminescent substrate.34 Light emis-sion was detected using a ChemiDoc™ XRS + System ImageLab™ Software (Bio-Rad, Hercules, CA). The bands were ana-lyzed using the ImageJ 1.42p digital imaging processing soft-ware (http://rsb.info.nih.gov/ij/March/27/2012).

2.3.9. Histological analysis. Samples of the liver, sub-cutaneous and visceral adipose tissue were dissected andimmediately fixed with ice-cold 4% (w/v) paraformaldehyde inphosphate bu!er saline (PBS) and embedded in para"n. Two4 µm sections per block were then stained with hematoxylinand eosin (H&E) and imaged at 20! magnification. Analysis ofthe adipocyte area was done using the Adiposoft software.35 Tovisualize neutral lipids, frozen liver sections (8 µm) werestained with oil Red O (Sigma St. Louis, MO). Morphologicalanalysis was performed using a Leica Qwin image analyzersystem on a Leica DMLS microscope (Wetzlar, Germany).

2.4. Statistical analysis

The results are presented as means ± standard error. Data wereanalyzed using the Graphpad Prism 6.0 software. The resultswere assessed by one-way ANOVA followed by Tukey’s test toidentify significant di!erences among the treatments. Di!er-ences between means were compared at a level of significanceof P < 0.05. Every experiment was performed in triplicate.

3. Results and discussion3.1. Characterization of Opuntia ficus-indica isorhamnetinglycoside extract

The extract contained isorhamnetin glycosides at a concen-tration similar to those previously reported.26 The OFI iso-rhamnetin glycoside extract is composed of isorhamnetin-glucosyl-rhamnosyl-rhamnoside (IG1), isorhamnetin-glucosyl-rhamnosyl-pentoside (IG2), isorhamnetin-hexose-methylpen-tose-pentose (IG3), isorhamnetin-glucosyl-pentoside (IG4), andisorhamnetin-glucosyl-rhamnoside (IG5) (Fig. 1).

3.2. E!ect of an OFI extract rich in isorhamnetin glycosideson body weight and metabolic parameters in a mouse modelof diet-induced obesity

To evaluate the metabolic e!ects of OFI isorhamnetin glyco-sides on the development of diet-induced obesity, we fed themice a high-fat (HF) diet alone or supplemented with 0.3% or0.6% OFI extract (low dose; LD or high dose; HD, respectively)for 12 weeks. Mice fed the HF diet showed an increased bodyweight compared to mice fed the control diet; interestinglythose receiving both doses of the OFI extract presented similarbody weights as the control mice (Table 2). It is important tonote that the changes in body weight were not due to adecrease in food intake since there was no significant di!er-ence in the daily energy intake in any group (Table 2). The OFI

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extract diminished the circulating total cholesterol, LDLcholesterol and HDL cholesterol in mice fed the HF diet, butdid not modify plasma triglycerides (Table 2). These results aresimilar to those reported using a co!ee extract containing fla-vonoids20 and also using quercetin glycosides from a cranberryextract.21 The HF-OFI fed mice had lower serum glucose andinsulin concentration than the HF fed mice. However, insulinconcentration tended to increase in the HF-OFI fed mice whencompared to control mice. As a result, HF-OFI fed miceshowed a significantly lower HOMA than the HF fed mice.Interestingly, there was no significant di!erence between theHF-OFI fed mice when compared to the control group.

3.3. The insulinotropic e!ect of an OFI extract rich inisorhamnetin glycosides in isolated pancreatic islets

To evaluate if the increased insulin concentration found inmice fed the OFI extract is a direct e!ect of OFI isorhamnetinglycosides on insulin secretion from pancreatic "-cells, we iso-lated rat pancreatic islets and evaluated the insulin secretionin response to the OFI isorhamnetin glycosides. The classical

pathway for insulin secretion from pancreatic "-cells is acti-vated in response to an increase in the GLUT2-mediatedglucose influx and its subsequent metabolism, increasing theATP/ADP ratio. Then, the ATP-sensitive potassium (KATP) chan-nels close and cause membrane depolarization that opensvoltage-dependent calcium channels resulting in insulin exoci-tosis.4 We found that the OFI extract tested at 2 #M isorhamne-tin equivalents, increased the insulin secretion compared tothe control (Fig. 2a) and to isolated isorhamnetin glycoside(IG1) or pure isorhamnetin (Iso). The lack of a significante!ect of IG1 and Iso on insulin secretion, indicates that othercomponents in the OFI extract besides IG1 or Iso are respon-sible for the insulinotropic e!ect on "-cells, as reportedpreviously by Chahdoura et al.23 and Antunes-Ricardo et al.36

However, when the extract was tested at 11 mM glucoseinstead of 5.5 mM, there was no statistically significant e!ecton insulin secretion when tested at 2 or 4 #M (Fig. S1, ESI†).These results suggest that the OFI extract is able to stimulatebasal insulin secretion, but it does not contribute to glucose-stimulated insulin secretion.

We found that the OFI extract increased GLUT2 mRNA inthe isolated pancreatic islets (Fig. 2b). It has been demon-strated that the expression of GLUT2 in the "-cells is regulatedby the transcription factor PPAR!.37,38 PPARs are members ofthe nuclear receptor superfamily of ligand-inducible transcrip-tion factors, and are a family containing three isoforms,PPAR%, PPAR! and PPAR"/&. PPAR! is mainly expressed inwhite adipose tissue, participating in the control of adipocytedi!erentiation and triglyceride esterification.39 This transcrip-tion factor is also involved in the control of the gene networkthat regulates glucose-stimulated insulin secretion in pancrea-tic "-cells, including the gene encoding for GLUT2, as hasbeen demonstrated in the mice lacking PPAR!.40 Interestingly,OFI isorhamnetin glycosides also increased the expression ofPPAR! (Fig. 2c), indicating that polyphenols from Opuntiaplants could up-regulate PPAR! transcriptional activity andincrease insulin secretion by augmenting glucose entry into

Fig. 1 The Opuntia !cus-indica extract contains "ve isorhamnetin gly-cosides. Chromatogram of the Opuntia !cus-indica extract.

Table 2 Body weight and fasting parameters of mice after 12 weeks of treatment with di!erent diets: control, high fat, high fat + low dose (HF +LD) and high fat + high dose (HF + HD)a

Control High fat HF + LD HF + HD

Body weight (g) 29.1 ± 1.5b 37.2 ± 1.9a 26.6 ± 1.9b 27.2 ± 1.8b

Food intake (g per day) 3.0 ± 0.6a 3.0 ± 0.5a 2.5 ± 0.5a 2.6 ± 0.8 a

Energy intake (kcal per day) 11.8 ± 2.2a 14.6 ± 2.6a 11.9 ± 2.3a 12.6 ± 3.7a

Fasting serum parameters: glucose metabolismGlucose (mg dL!1) 317.4 ± 25b 370.2 ± 50a 217.6 ± 47c 304.5 ± 60b

Insulin (ng mL!1) 1.23 ± 0.4 4.49 ± 1.8a 1.48 ± 0.8b 1.76 ± 0.9b

Glucagon (pg mL!1) 96.5 ± 2.7a 81.5 ± 3.2b 61.3 ± 0.4d 70.8 ± 2.1c

HOMA-IR 23.86 ± 3.4b 101.59 ± 9.2a 19.65 ± 4.5c 32.87 ± 5.7b

Fasting serum parameters: lipid metabolismTotal cholesterol (mg dL!1) 94.0 ± 6.2c 129.3 ± 5.1a 121.6 ± 8.6a,b 104.9 ± 0.3b

LDL – cholesterol (mg dL!1) 9.6 ± 0.3b 16.3 ± 2.0a 16.7 ± 1.5a 11.0 ± 1.9b

HDL – cholesterol (mg dL!1) 83.4 ± 6.0c 119.7 ± 5.1a 114.5 ± 8.0a,b 102.1 ± 3.4b

Triglycerides (mg dL!1) 77.3 ± 1.3a 85.2 ± 7.8a 74.2 ± 9.2a 80.1 ± 6.6a

Leptin (ng mL!1) 3.3 ± 0.9c 15.1 ± 0.4a 8.1 ± 0.5b 7.3 ± 1.1b

a Every experiment was performed in triplicate (n = 3). Data are expressed as means ± SE. Rows with di!erent letters indicate significantdi!erences, P < 0.05.

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the "-cell through GLUT2. This suggestion is supported byother studies that have demonstrated that isorhamnetin,41 orquercetin can modulate PPAR! activity,42 whereas other poly-phenols as the soy isoflavone genistein, exert the oppositee!ect; i.e., reducing the PPAR! expression and insulinsecretion in pancreatic islets.29

3.4. An OFI extract rich in isorhamnetin glycoside improvesglucose tolerance in a mouse model of diet-induced obesity

Mice fed the HF diet for 12 weeks presented glucose intoler-ance as demonstrated by the increase in the AUC of an ipGTTcompared to those fed the control diet (Fig. 3a). Interestingly,mice fed the HF diet supplemented with a low dose of the OFIextract showed improved glucose tolerance. The ipGTT AUC ofHF + LD mice was similar to that of mice fed the control diet,whereas those fed the HF + HD presented an AUC similar tothat of the HF group (Fig. 3a). A similar response was pre-viously reported with an extract rich in quercetin.21 Our datasuggest that the OFI extract improves glucose intolerance in adose-dependent manner.

3.5. An OFI extract rich in isorhamnetin enhances energyexpenditure in a mouse model of diet-induced obesity

To evaluate if the reduction in body weight observed in micefed the OFI extract was a result of increased metabolic rate, weassessed energy expenditure by indirect calorimetry. Interest-ingly, we found that both doses of isorhamnetin glycosidescaused an increase in oxygen consumption (Fig. 3b), indicat-ing enhanced energy expenditure. An HF diet induces meta-

bolic inflexibility, decreasing the whole-body capacity tooxidize carbohydrates during the feeding state, relying only onlipid oxidation.43 Metabolic inflexibility is typically recognizedwith an RER of 0.7 during the feeding period (0% oxidation ofcarbohydrates vs. 100% oxidation of fat)43 (Fig. 3c). Interest-ingly, mice that consumed isorhamnetin glycosides presentedan RER of 0.8 (32% oxidation of carbohydrates vs. 68% oxi-dation of fat), significantly higher compared to mice fed theHF diet alone (Fig. 3c). This result is in accordance with pre-vious reports using other polyphenols such as resveratrol44

and catechin polyphenols of green tea.45 These results indicatethat the OFI extract increases energy expenditure and modifiesthe type of substrates used, suggesting an improvement in themetabolic flexibility.

3.6. An OFI extract prevented adipose tissue hypertrophy andreduced leptin concentration in mice fed an HF diet

Adipose tissue hypertrophy, lipid spillover and hyperleptine-mia are thought to be the earliest signs of metabolic altera-tions during obesity,46,47 leading to hepatic and skeletalmuscle lipid accumulation and lipotoxicity.46 Thus, we thenevaluated the e!ect of the OFI extract on adipose tissue mor-phology. As observed in Fig. 4, mice fed the HF diet showed anincrease in adipocyte size in both subcutaneous (Fig. 4a) andvisceral (Fig. 4b) adipose tissue depots with respect to thosefed the control diet. Interestingly, mice supplemented with iso-rhamnetin glycosides exhibited a decrease in adipocyte size inboth the adipose tissue depots, despite being on an HF diet.Adipocyte size presented a significant correlation with serum

Fig. 2 The Opuntia !cus-indica extract increases in vitro insulin secretion. (a) Insulin secretion in rat pancreatic islets Ext: Opuntia !cus-indicaextract, IG1: isorhamnetin-glucosyl-rhamnosyl-rhamnoside, Iso: isorhamnetin tested at 2 #M isorhamnetin equivalents. Real time RT-PCR for (b)GLUT2 (glucose transporter type 2), and (c) PPAR! (peroxisome proliferator-activated receptor gamma). Every experiment was performed in tripli-cate (n = 3). Data are expressed as means ± SE. Bars with di!erent letters indicate signi"cant di!erences, P < 0.05.

Fig. 3 E!ect of Opuntia !cus-indica extract on glucose tolerance and energy expenditure. Glucose tolerance test and indirect calorimetry in mice(n = 5 for each group) after 10 and 11 weeks respectively of treatment with control diet, high fat diet , and high fat diet enriched with Opuntia !cus-indica extract at low (HF + LD) and at high (HF + HD) doses (a) postprandial oxygen volume (VO2) consumption (b) and postprandial RER (c). Data areexpressed as means ± SE. Bars with di!erent letters indicate signi"cant di!erences, P < 0.05.

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leptin content in the four groups (Fig. 4c). Leptin is a proteinhormone secreted by adipocytes that regulates food intake andenergy expenditure to maintain body fat stores. Then, if thebody fat mass increases, plasma leptin concentration increasesto the same extent. As expected, circulating leptin concen-tration in mice fed the HF diet was higher than that observedfor mice fed the control diet. Interestingly, circulating leptin inmice fed the HF diet supplemented with an OFI extract waslower than in HF diet fed mice and this corresponded to thelower body weight observed (Table 2). These results indicatethat OFI isorhamnetin glycosides prevent adipose tissue hyper-trophy and hyperleptinemia in mice fed an HF diet. It isimportant to note that the OFI extract used here has 0.0087%isorhamnetin equivalents, and in a previous report, the admin-istration of a 0.3% pure quercetin extract (a dose much higherthan the dose used in this study), did not cause significantreduction in body weight or adipose tissue sizes.11 Thus, ourdata suggest that the use of a lower dose of the OFI extract ismore e!ective in preventing adipose tissue hypertrophy.

3.7. An OFI extract rich in isorhamnetin glycoside improveshepatic insulin signaling in mice fed an HF diet

Insulin resistance in the liver is a frequent metabolic alterationassociated with defective insulin signaling observed duringdiet-induced obesity.48 To evaluate the e!ect of the OFI extracton hepatic insulin signaling, we determined the IRS1 and AKTmRNA abundance and protein levels, as well as the phos-phorylation state of IRS1 Tyr941 and the mTORC1 target S6 KThr389, two proteins of the insulin-activated signalingcascade.49 Insulin binds to its receptor increasing the tyrosinekinase activity and phosphorylation of IRS1. The Tyrosine 941

phosphorylation of IRS1 leads to the activation of phosphoino-sitide-dependent protein kinase and downstream signal trans-duction intermediates such as AKT.50 We found that themRNA expression of IRS1 and AKT in the liver presented a ten-dency to increase when supplemented with either dose of theisorhamnetin glycosides, but the di!erence did not reach stat-istical significance (Fig. 5a and 5b). Interestingly, IRS1 proteinabundance decreased with HF diets even in the presence ofthe OFI extract. Nonetheless, we found that isorhamnetin gly-cosides increased IRS1 Tyr941phosphorylation despite thereduction in the total IRS1 protein observed in all groupsreceiving the HF diet (Fig. 5c and 5d).

In addition, it is known that the activation of the insulinsignaling cascade upregulates the mammalian target of rapa-mycin complex 1 (mTORC1) leading to the phosphorylation ofS6 K Thr-389.49 We observed an increase in S6 K Thr389 phos-phorylation in mice supplemented with the OFI extract,despite the reduced abundance of total S6 K in the liver(Fig. 5e and 5f). These results indicate an enhanced insulinsignal transduction in the liver of mice receiving isorhamnetinglycosides through the HF diet.

3.8. An OFI extract reduces hepatic steatosis and amelioratesendoplasmic reticulum stress in a mouse model ofdiet-induced obesity

Hepatic steatosis is one of the most prevalent conditionsassociated with obesity and insulin resistance, and it can pro-gress to more severe hepatic diseases such as nonalcoholicsteatohepatitis, hepatic fibrosis and hepatocarcinoma.51 Micereceiving the high-fat diet developed fatty liver as determinedby H&E and oil red O staining (Fig. 6a), primarily due to trigly-

Fig. 4 Opuntia !cus-indica extract diminished adipose cell size and leptin secretion. Hematoxylin and Eosin (H&E) stain for subcutaneous and vis-ceral adipose tissue and size distribution of adipocytes in subcutaneous (a) and visceral adipose tissue (b). Leptin secretion and adipocyte cellsize (c). Data are expressed as means ± SE. Bars with di!erent letters indicate signi"cant di!erences, P < 0.05.

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cerides rather than cholesterol accumulation (Fig. 6b and 6c).Interestingly, isorhamnetin glycosides reduced hepatic lipidvesicles and triglyceride content. To better understand the

molecular mechanisms involved in the lipid-lowering e!ectof the isorhamnetin glycosides, we measured several genesinvolved in the hepatic lipid metabolism. The transcription

Fig. 5 The Opuntia !cus-indica extract increased insulin signaling in the liver in vivo. Real time RT-PCR for (a) IRS1, and (b) AKT in the liver. Phos-phorylated (tyr941) of IRS-1 (c) total IRS1 (d), Phosphorylated (thr389) S6 K (e) and total S6 K (f ) as a!ected by the dietary administration of an OFIextract with isorhamnetin glycosides. Every experiment was performed in triplicate (n = 3). Data are expressed as means ± SE. Bars with di!erentletters indicate signi"cant di!erences, P < 0.05.

Fig. 6 The Opuntia !cus-indica extract diminished hepatic lipid accumulation. Hematoxilin and eosin and oil red O staining of liver sections (a).Total cholesterol (b) and total triglycerides content (c) in the liver. Every experiment was performed in triplicate (n = 3). Data are expressed as means± SE. Bars with di!erent letters indicate signi"cant di!erences, P < 0.05.

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factor SREBP1 regulates various genes that contribute to fattyacid synthesis and esterification.52 It has recently beenobserved that the activation of endoplasmic reticulum stressincreases SREBP1 transcriptional activity, inducing lipid syn-thesis and triglyceride accumulation in the liver.53 Mice receiv-ing the high fat diet presented an increase in the mRNAcontent of several endoplasmic reticulum stress markers in theliver, such as ATF6, XBP1 and CHOP (Fig. 7a–c). Interestingly,an OFI extract with isorhamnetin glycosides prevented theinduction of these genes along with a reduced mRNAexpression of SREBP1, FAS and SCD1 (Fig. 7d–f ). This issimilar to a previous study where it was found that resveratroland pterostilbene diminished the expression of FAS and par-tially prevented the increase in body weight due to the con-sumption of a high fat diet.54 It has been demonstrated thatcertain molecules can act as synthetic chaperones, preventingendoplasmic reticulum stress.55 Thus, the present resultssuggest that the OFI isorhamnetin glycoside extract maycontain molecules that can act as molecular chaperones, ame-liorating endoplasmic reticulum stress, reducing SREBP1 acti-vation and lipid accumulation in the liver.

Although the expression of PPAR% was not significantlydi!erent among treatments (Fig. 7g), there was a significantincrease in the expression of CPT1 and AOX, two proteinsinvolved in mitochondrial fatty acid oxidation in mice sup-

plemented with an OFI extract with isorhamnetin glycosides(Fig. 7h and 7i). These results suggest that more fatty acidswere entering the mitochondria for oxidation, which is in

Fig. 7 The Opuntia !cus-indica extract diminished endoplasmic reticulum stress in the liver and genes involved in lipogenesis. Real time RT-PCRfor ATF6 (a), XBP1 (b), CHOP (c), SREBP1 (d), FAS (e), SCD1 (f ), PPAR% (g), CPT1 (h) and AOX (i) in liver. Every experiment was performed in triplicate(n = 3). Data are expressed as means ± SE. Bars with di!erent letters indicate signi"cant di!erences, P < 0.05.

Fig. 8 Proposed mechanisms underlying the reduction of body weightgain due to the dietary administration of the Opuntia !cus-indica extractin a mouse model of diet-induced obesity.

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agreement with the major energy expenditure observed inthese groups. These metabolic e!ects were reflected in theoverall fat reduction in mice supplemented with isorhamnetinglycosides.

4. ConclusionThe OFI isorhamnetin glycoside extract reduces body weightgain, increases insulin secretion and increases energy expendi-ture in the mice fed a high-fat diet. These metabolic e!ectstogether with the lower fatty acid synthesis and higher fattyacid oxidation caused a decrease in hepatic and adipose tissuefat accumulation preventing hepatic steatosis and adipocytehypertrophy. Therefore obesity was prevented by the dietaryadministration of the OFI isorhamnetin glycoside extract(Fig. 8).

Con#ict of interestNone.

AcknowledgementsThis research was supported by the Research Chair FundsCAT-005 from Tecnológico de Monterrey Campus Monterrey,the scholarship (CONACYT CVU-295380) of the first author,the nopal project Consejo Nacional de Ciencia y Tecnología(CONACYT-CB 168708) and Nutrigenomic FEMSA ResearchFund.

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