Phenolic-enriched raspberry fruit extract (Rubus idaeus) resulted in lower weight gain, increased ambulatory activity, and elevated hepatic lipoprotein lipase and heme oxygenase-1 expression in male mice fed a high-fat diet.

Abstract

Red raspberries (Rubus idaeus) contain numerous phenolic compounds with purported health benefits. Raspberry ketone (4-(4-hydroxyphenyl)-2-butanone), is a primary raspberry flavor phenolic found in raspberries and is designated as a synthetic flavoring agent by the FDA. Synthetic raspberry ketone has been demonstrated to result in weight loss in rodents. We tested whether phenolic-enriched raspberry extracts, compared with raspberry ketone, would be more resilient to the metabolic alterations caused by an obesogenic diet. Male C57BL/6J mice (8 weeks old) received a daily oral dose of vehicle (VEH; 50% propylene glycol, 40% water, and 10% DMSO), raspberry extract low (REL; 0.2 g/kg), raspberry extract high (REH; 2 g/kg) or raspberry ketone (RK; 0.2 g/kg). Coincident with daily dosing, mice were placed on a high-fat diet (45% fat kcal). After 4 weeks, REH and RK reduced body weight gain (approximately 5-9%) and white adipose mass (approximately 20%) compared with VEH. Hepatic gene expression of heme oxygenase-1 and lipoprotein lipase were upregulated in REH compared with VEH. Indirect calorimetry indicated that respiratory exchange ratio (RER; v.CO2/v.O2) was lower, suggesting increased fat oxidation with all treatments. REH treatment increased total ambulatory behavior. Energy expenditure/lean mass was higher in REH compared with REL treatment. There were no treatment differences in cumulative intake, meal patterns, or hypothalamic feed-related gene expression. Our results suggest that raspberry ketone and a phenolic-enriched raspberry extract, both have the capacity to prevent weight gain, but differ in the preventative mechanisms for excess fat accumulation following high-fat diet exposure.

1. Introduction

Red raspberry (Rubus idaeus) is a popular consumer fruit grown mostly in northern temperate regions. In addition to their taste and nutritious appeal, red raspberries are abundant in bioactive phytochemicals [1]. In particular, red raspberries have a relatively high level of anthocyanins and are rich in hydrolysable tannins of ellagitannins compared with other fruits [2]. Whole fruit or raspberry extracts have been examined for their therapeutic potential for improving markers for several diseases and pathological conditions [3, 4]. With regards to metabolic diseases, raspberries or raspberry-containing berry combinations have been shown to reduce adiposity in rodent models of diet-induced obesity [5] and reduce lipid accumulation in vitro [6]. Although whole raspberry fruit acts, in part, through several metabolic pathways, such as AMP-activated protein kinase (AMPK) and NLR family pyrin domain containing 3 (NLRP3) inflammasome [7, 8], a more complete examination of the bioactive phytochemicals of raspberries is needed to fully understand the therapeutic potential of red raspberries for treating obesity and related metabolic diseases.

Phenolics, including polyphenols and ellagitannins, make up a larger percentage of the bioactive phytochemicals in red raspberries [9]. One characteristic phenolic derived from raspberries is 4(4-hydroxyphenyl)-2-butanone, also known as frambinone or simply raspberry ketone [10]. Raspberry ketone is considered the principal aroma and flavor phenolic of raspberries [11, 12]. Fully ripened raspberries contain trace amounts of raspberry ketone. As such, most commercially available raspberry ketone is derived from synthetic synthesis. In 1965, raspberry ketone was designated as a generally recognized as safe (GRAS) food additive and is listed as a synthetic flavoring substance/adjuvant by the U.S. Food and Drug Administration (FDA)[13]. Based on the structural similarity to other bioactive phenolic compounds, however, raspberry ketone has been examined for anti-obesity potential in rodents and in in vitro systems [8, 14–19]. In vivo rodent studies suggest that raspberry ketone may be effective at preventing the metabolic alterations associated with excessive caloric intake, rather than reversing the metabolic effects caused by obesity [16, 17, 20]. The preventative capacity of raspberry ketone is supported by in vitro studies demonstrating that raspberry ketone acts by inhibiting expression of genes related to adipogenesis, to prevent adipocyte differentiation and lipid accumulation [14, 15, 18, 19, 21].

Red raspberries have been shown to be rich in other phenolic compounds (e.g., flavonoids, phenolic acids, tannins) and whole fruit extract have anti-hypertensive, antiinflammatory, and cancer preventative capacities [22–24]. One underexplored concept is that the other naturally derived phenolic abundant derivatives of red raspberries can improve metabolic resilience in an obesogenic environment. The purpose of this study was to whether a phenolic-enriched raspberry fruit extract has a potential to prevent the excess weight gain and metabolic alterations associated with the development of diet-induced obesity. Our hypothesis was that the phenolic-enriched raspberry extract would prevent diet-induced excessive weight gain. The anti-obesity potential of the phenolic-enriched raspberry extract, which did not contain any detectable levels of naturally-derived raspberry ketone, was compared with synthetic raspberry ketone. Because the in vivo preventative mechanism of action of raspberry ketone are not fully known, our comparison included examining excess body weight and fat gain, meal patterns, glucose homeostasis and endocrine markers, energy expenditure, and feeding-related hypothalamic and metabolic-related hepatic gene expression alterations induced by high-fat diet consumption.

2. Methods and Materials

2.1. Raspberry extract source and phenolic enrichment procedure

Raspberry extracts were prepared from a commercially available whole-fruit red raspberry juice concentrate (FruitFast; www.brownwoodacres.com). According to the juice concentrate supplier, red raspberries were sourced from the Pacific Northwest region of the United States. The raspberry juice concentrate (per 1 oz) had 1 g of total fat (0 g saturated fat, 0 g trans fat), 15 g of total carbohydrates (0 g dietary fiber, 13 g sugar). Preparation of phenolic-enriched extract was performed similar to the enrichment methods described elsewhere [25, 26]. In brief, the fruit concentrate was acidified with 1 % acetic acid and filtered through Whatman filter paper #4 with the aid of suction. Filtrate of the acidified extract was portioned against ethyl acetate (EtOAc) to remove lipophilic materials. After evaporation of any remaining EtOAc, the aqueous layer was loaded on an Amberlite XAD (Sigma-Aldrich, St. Louis, MO) and a preconditioned 1% acetic acid column. The resin was washed thoroughly with 1% acetic acid to remove sugars and phenolic acids. The phenolic mixture was eluted with methanol, evaporated, and freeze-dried. A reference sample (~1 g) from each batch and raspberry ketone (4[4-hydroxyphenyl]-2-butanone; 99%; cat#178519; Sigma Aldrich) were deposited in a secure, climate-controlled repository [28].

2.2. Phenolic-enriched red raspberry extract analyses

A freeze-dried raspberry extract sample (36.9 mg) was prepared in 25 ml of 70% methanol acidified with 0.1% formic acid, vigorously vortexed, and then sonicated for 5 min. The extract was then filtered through 0.45 μm filter prior to polyphenol analysis by HPLC-UV/vis-MS (Agilent 1100 series; LC/MSD trap, Waldbronn, Germany). The HPLC was equipped with an autodegasser, quaternary pump, autosampler, column thermostat, and diode array detector (DAD). The column used for compound separation was Agilent Polaris 3 Amide-C18, 250 × 4.6 3μm, 3pm (Santa Clara, CA). The mobile phase A was water modified with 0.2 % trifluoroacetic acid (TFA; Fisher Scientific; Fair Lawn, NJ) and mobile phase B was acetonitrile with 0.2% TFA. The flow rate was 1 mL/min. The gradient was 10 to 15% B from 0 to 7 min, 15 to 25% from 7 to 30 min, and 25 to 40% from 30 to 45 min. The column was equilibrated with 10% B for 10 min between injections and thermostatted at 25 °C with an injection volume of 5 μL. The DAD was set at 260 nm, 370 nm, and 520 nm, with a spectrum scan range from 200 to 600 nm. The MS featured an electrospray ionization (ESI) source and ion trap mass analyzer. The nebulizer was set at 40 psi and drying gas at 350 °C with its flow rate at 12 L/min. Nitrogen was used as a nebulizing and a drying gas. For the positive scan, the collision energy termed as compound stability was set at 60% and the scan range was 200 to 1200 m/z. For negative polarity, collision energy was set at 120% with a scan range of 200 to 2200 m/z. Helium was used as the collision gas for both scan polarities. Positive and negative scans were conducted in separate runs.

Anthocyanins were quantified using cyanidin chloride (ChromaDex, Inc., Irvine, CA) as the external standard and corrected by the corresponding molecular weight against the reference standard. Hydrolyzable tannins were quantified as total ellagic acid (Sigma-Aldrich: St. Louis, MO) after hydrolysis using a method described elsewhere [27]. Briefly, 10 mL raspberry extract solution was mixed with 1.8 mL TFA to make a final solution of 2 M TFA and refluxed for 2 h. After reacted solvent was cooled and brought to a final volume of 25 mL, the solvent was filtered before for LC/UV/MS analysis. The percentage of polyphenolic content were determined by using Folin & Ciocalteu’s reagent (Sigma- Aldrich, St. Louis, MO). Serial concentrations of gallic acid were used as the calibration. The total polyphenol content in the raspberry extract was presented as the equivalent gallic acid content.

2.3. Raspberry ketone quantification in raspberry extract

The diluted (10X) sample of raspberry extract that was prepared for polyphenol determination was used to quantify raspberry ketone by ultra-high-performance liquid chromatography (UHPLC; Agilent 1290 Infinity II) with tandem mass spectrometry (MS/MS methods; Agilent 640 triple quadrupole). The column used for separation was Waters Acquity BEH C18 column, 50 × 2.1 mm, 1.7 μm (Milford, MA). The mobile phase A was water with 0.1% acetic acid and mobile phase B was acetonitrile with 0.1% acetic acid. The flow rate was 0.4 mL/min. The gradient was isocratic elution at 28% B and the injection volume was 4μL. The ESI settings and MS/MS transitions were optimized for raspberry ketone detection. The instrumental limit of detection (LOD) for RK was 1.72 ng/mL, which corresponded to a LOD of 11.6 ng RK/mg raspberry extract or 0.0012%[28].

2.4. Mice

Male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were fed standard chow (Purina Mouse Diet 5015, 25.34% fat, 19.81% protein, 54.86% CHO, 3.7 Kcal/g) and water was available at all times, unless otherwise noted. Mice were pair-housed, unless otherwise noted, and maintained on a 12 h light, 12 h dark cycle with lights on from 0700 h to 1900 h. The animal care protocol was approved by the Institutional animal Care and Use Committee of Rutgers University (OLAW #A3262-01, protocol #13-001).

2.5. Phenolic-enriched raspberry extract pretreatment and oral glucose tolerance test (OGTT)

An extract tolerance test, and oral glucose tolerance with pretreatment was performed in a separate cohort of mice (12 weeks old; n = 10). Each mouse was food deprived for 5 h. Oral dosing was performed using single-use, sterile plastic feeding tubes (20 ga × 30 mm; cat# FTP-20-30, Instech Laboratories, Plymouth Meeting, PA). In a randomized order, each mouse was orally dosed with vehicle (VEH; 50% propylene glycol, 40% water, and 10% DMSO), raspberry extract high dose (REH; 2 g/kg), raspberry extract low dose (REL; 0.2 g/kg), and raspberry ketone (RK; 0.2 g/kg). The doses of the phenolic-enriched raspberry extract were based on the maximum solubility in the vehicle at 2 g/kg. Raspberry ketone doses were based on preliminary studies demonstrating acute feeding suppression at 0.2 g/kg. Mice received each dose with at least a 7-day washout between doses. At the start of the test, mice were placed in plastic restrainers, and a tail nick was performed to obtain a baseline glucose level (0 min) using a glucometer (AlphaTRAK 2, Zoetis, Kalamazoo, MI). Subsequent readings were taken at 15 min, 30 min, 60 min, 90 min, 120 min, and 180 min following dosing. For the extract glucose tolerance test, tail blood glucose was measured immediately after oral dosing. For the pretreatment OGTT, mice were orally dosed with vehicle, REH, REL, and RK at 60 min before being orally dosed with glucose (2 g/kg).

2.6. High-fat diet feeding, dosing groups, and terminal measurements

Mice (8 weeks old) were equally divided by body weight and were switched to a high-fat diet (HFD; 4.73 kcal/g, 45% fat, 20% protein, 35% carbohydrate; D12451; Research Diets, Inc., New Brunswick, NJ; Table 1) fed ad libitum. Coincident with the diet switch, daily oral dosing was initiated in mice with the following treatments: VEH (n = 23), REH (n = 24), REL (n = 24), or RK (n = 24). Mice were fed the high-fat diet and orally dosed for 4 weeks. Daily body weight and cumulative food intake were measured throughout the entire dosing period. Daily dosing was performed between 1000 h and 1200 h. At the completion of dosing, an OGTT was performed following a 5-h calorie deprivation. To quantify body fat, a midabdominal incision was performed on each carcass to identify and remove retroperitoneal, epididymal, and subcutaneous (inguinal) fat pads. The collected fat pads were weighed to the nearest 0.01g.

Table 1.

Ingredient composition of the high-fat (45% Fat Kcal) diet fed to mice^a

Ingredient	g/kg
Casein, Lactic, 30 mesh	233.1
Cystine, L	3.5
Sucrose	206.0
Lodex 10 Maltodextrin	116.5
Corn Starch	84.8
Fiber Solka Floc, FCC200	58.3
Lard	206.8
Soybean Oil, USP	29.1
Mineral mix S10026B	58.3
Choline Bitartrate	2.3
Vitamin mix, V10001C	1.2
Dye, Red FD&C #40, Alum. Lake 35–42%	0.058
Total	1000

^aD12451; Research Diets, Inc., New Brunswick, NJ

2.7. Meal patterns and meal microstructures

Meal microstructures were analyzed using the Biological Data Acquisition System (BioDAQ; Research Diets, New Brunswick, NJ). This system utilizes standard shoe-box style cages with a gated front-mounted food hopper. Mice were acclimated to the cages for 4 days to stabilize feeding patterns before recording the experimental data over 3 days. Mice were housed in the BioDAQ system for a total of 7 days. The gated hopper sits upon a sensor that detects net changes in food weight per second. A feeding bout was defined as a change in stable weight of the hopper. Bouts were clustered into meals, defined by an inter-meal interval of 300 sec and a minimum of 0.02 g consumed [29]. Experimental data were calculated for 3 days (3 dark and 3 light periods). Meal patterns were measured over the 72 h while groups were maintained on their respective dosing protocols. Data were expressed as bouts, meal frequency (or meal number), average meal size (mg/meal), meal duration (duration of meals/meal number), meal eating rate (mg/min), and total daily meal intake (g) averaged for the 24-h period.

2.8. Body composition and respiratory exchange ratio (RER)

Body composition was assessed using the EchoMRI 3-in-1 body composition analyzer (Echo Medical Systems, Houston, TX). The Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH), an indirect calorimeter, was used to measure O₂ consumption (v.O₂), CO₂ production (v.CO₂), and RER (v.CO₂/v.O₂) at 25°C. Energy expenditure was estimated according to CLAMS energy equation; EE (Cal/h) = [3.815 × v. CO₂ + 1.232 × V.O₂]. Ambulatory activity was also measured by an activity monitor using IR photocells to calculate activity. Mice were maintained on their respective dosing protocols and housed in the system for 7 days. The last 24-h epoch (dosing Day 28) was used for the analysis [30].

2.9. Terminal plasma hormones and blood glucose

After 4 weeks of dosing, mice received a 5-h calorie deprivation before euthanasia by decapitation. Final respective oral treatments were 24 h prior to euthanasia by decapitation. Blood was collected in K2 EDTA tubes and with an added protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (1 mg/mL; Sigma-Aldrich, St. Louis, MO). Samples were maintained on ice until centrifugation at 3000 rpm for 10 min at 4 °C. Plasma was stored at −80 °C until analysis. The mouse metabolic hormone magnetic bead kit (cat # MMHMAG-44K; EMD Millipore) for the Luminex xMAP was used for the simultaneous quantification of plasma hormones in each sample. The multiplex kit measured ghrelin (active; sensitivity: 7 pg/ml), insulin (sensitivity: 69 pg/ml), and leptin (sensitivity: 69 pg/ml). A separate singleplex bead kit was used to measure adiponectin (cat # MADPNMAG-70K). A radioimmunoassay (RIA) kit was used to determine plasma levels of corticosterone (cat # 07120103, MP Biomedicals; sensitivity: 25 ng/mL). Degree of insulin resistance was estimated by homeostasis assessment model (HOMA-IR), calculated as follows: HOMA-IR = [fasting serum glucose (mmol/L) × fasting serum insulin (mU/L)] / 22.5. Insulin values were expressed as (1 IU = 0.04167 mg) [31, 32].

2.10. Tissue dissection for quantitative real-time PCR (qPCR), RNA extraction, and RT-qPCR

Immediately following euthanasia by decapitation, the mice liver and brain were excised. The liver and brain were washed in ice cold Sorensen’s buffer. It was then fixed in RNA later (ThermoFisher Scientific, Inc.), and stored at −80C. The brain was cut into 1 mm coronal blocks using a brain matrix (Ted Pella, Redding, California), anterior (bregma: −0.70 to −1.34 mm) and posterior (bregma: −1.35 to −1.94 mm). The brain blocks were stored in RNA later overnight at 4 °C. The arcuate and paraventricular nuclei were dissected from the slices using a dissecting microscope and stored at −80 °C. Liver RNA was extracted using a standard TRIzol extraction coupled with Machery-Nagel Nucleospin RNA extraction kit with DNase I treatment. Total RNA from the arcuate (ARC) and paraventricular (PVN) nuclei were extracted using the Ambion RNAqueous Micro kits (Invitrogen) as per manufacturer’s protocol. Total RNA was treated with DNase-Ι using the extraction kit protocol at 37 °C for 30 min to minimize any genomic DNA contamination. Liver, ARC, and PVN RNA quantity and quality was determined using a NanoDrop ND-2000 spectrophotometer (ThermoFisher, Waltham, Massachusetts) and an Agilent 2100 Bioanalyzer and RNA Nano Chips (Agilent Technologies, Santa Clara, California). Only samples with RNA Integrity Number (RIN)>8 were used. Reverse transcription was done on 200 ng of total RNA using Superscript III reverse transcriptase (ThermoFisher Scientific, Inc.), 4 μl 5× SS buffer, 1.25 μl 100mM dNTP, 100ng random hex primers (Promega Corp), 40 U/μL Rnasin (Promega), and 100 mM dithiothreitol in DEPC-water in a total volume of 20 μl. Reverse transcription was done using the following protocol: incubation at 25 °C for five minutes, transcription at 50 °C for 60 min, denaturation at 70 °C for 15 min and then cooled to 4 °C for five minutes. The cDNA was diluted 1:20 using nuclease free water for a final cDNA concentration of 0.5ng/μl and stored at −80 °C. Naive hypothalamus and liver tissue RNA was used for positive and negative controls (no reverse transcriptase) and processed simultaneously with experimental samples.

All PrimePCR™ primers were purchased from BioRad Laboratories, Inc. and are commercially available. These primers included Hmox1, Lipe, Lpl, Rpl30. Primers for following genes were synthesized by Life Technologies, Inc.: Adra1a, Adra2c, Agrp, Cart, Crh, Fas, Gapdh, Hprt, Npy, Pgc1a, Pomc, Ppara, Pparg, Scd1, and Trh. see supplemental Table S1 for primer sequences. qPCR was performed on 4 μl of 0.5ng/μl of cDNA using SsoAdvanced^Tm Universal SYBR® Green Supermix (Bio Rad Laboratories, Inc.), primers, and nuclease free water. Relative mRNA expression was calculated using the δδC_T method. Geometric means of the reference genes Gapdh and Hprt was used to calculate δC_T for ARC and PVN samples, and geometric means of the reference genes Gapdh and Rpl30 was used to calculate δC_T for liver samples. A CFX-Connect real-time PCR instrument (Bio Rad) was used to amplify the samples according to the following protocol: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10s, annealing at 60 °C for 45s, and completed with a dissociation step for melting point analysis with 60 cycles of 95 °C for 10s, 65 °C to 95 °C (in increments of 0.5°C) for 5s and 95 °C for 5s. Quantification values were generated only from samples showing a single product at the expected melting point [30, 33].

2.11. Statistical Analyses

Data are presented as means ± SEM. Separate Analysis of variance (ANOVA) or ANOVA with repeated measures were performed to determine the effects of treatment conditions on individual measured parameters. When justified, Newman-Keuls post-hoc tests were performed unless otherwise specified. All statistical and power analyses were performed using Statistica 7.1 software (StatSoft) and significance was set at α = 0.05.

3. Results

3.1. Phenolic-enriched raspberry extract analyses and raspberry ketone determination

The chemical profile of the phenolic-enriched extract indicated that the major polyphenols were anthocyanins and ellagitannins. The predominant anthocyanins were glycosylated cyanidins, whereas the predominant ellagitannin was sanguiin-H6, see supplemental Fig S1 and Table S2. The gallic acid equivalent (GAE) total polyphenol content in the phenolic-enriched raspberry extract was 37.6%. Raspberry ketone was not detected in the phenolic-enriched raspberry extract.

3.2. Phenolic-enriched raspberry extract pretreatment and OGTT

An extract tolerance was performed to determine whether the phenolic-enriched extracts and raspberry ketone altered blood glucose levels, see supplemental Fig S2. For the extract tolerance test there was a time effect [F (6, 54) = 30.6, p < 0.0001]. Post-hoc tests indicated that blood glucose values returned to baseline at 60 min. However, neither doses of extract nor RK changed blood glucose values or incremental area under the curve (iAUC), see Fig.1A and inset. To determine whether the compounds acutely improved glucose tolerance, an OGTT was performed following a pretreatment. Because we observed a return to blood glucose to baseline levels 60 min after extract dosing, pretreatment with the extract and RK dosing was done 60 min prior to the OGTT. For the OGTT, there was a pretreatment effect [F (3, 27) = 4.3, p < 0.05] and time effect [F (6, 54) = 114.1, p < 0.0001]. Post-hoc testing indicated REH treatment had higher blood glucose than REL treatment (p < 0.01). At 30 min, there was higher blood glucose with REH compared with VEH and REL treatments (p < 0.05). At 60 min, the REH treatment resulted in higher blood glucose than REL treatment (p< 0.05), see Fig. 1B. For iAUC, there was also an effect [F (3, 27) = 4.13, p < 0.05] with REH treatment having higher iAUC compared with REL treatment (p < 0.05), see Fig. 1B inset.

Fig. 1.

Effects of pretreatment of phenolic-enriched raspberry extract on blood glucose and oral glucose tolerance test (OGTT). Each chow-fed male C57BL/6J mouse (12 weeks; n = 10) received an oral dose of vehicle (50% propylene glycol, 40% water, and 10% DMSO), raspberry extract low dose (REL; 0.2 g/kg), raspberry extract high dose (REH; 2 g/kg), and raspberry ketone (0.2 g/kg) treatment. There was at least a 7-day washout period between treatment and doses were administered in a randomized fashion. Data are represented as means ± SEM. A: Blood glucose immediately following oral dosing, data were analyzed with a repeated measures ANOVA. Inset: Incremental area under the curve (iAUC) of blood glucose following oral dosing, data were analyzed with a one-way ANOVA. B: OGTT following a 60-min pretreatment with the respective compound, data were analyzed with a repeated measures ANOVA. Inset: iAUC of the OGTT following a 60-min pretreatment with respective compound. Data were analyzed with a one-way ANOVA. For each analysis, Newman-Keuls post-hoc testing was performed when justified, * indicates p < 0.05 from vehicle treatment, # indicates p < 0.05 from REL treatment.

3.4. Preventative effects of phenolic-enriched raspberry extract on body weight gain and white adipose tissue gain associated with high-fat diet feeding

Daily dosing of treatments was initiated with the availability of the high-fat diet, see supplemental Fig S3. For daily body weights over the 28-day dosing period, there were treatment group [F (3, 59) = 3.3, p < 0.05], time [F (27, 1593) = 131.9, p < 0.0005], and treatment × time [F (81, 1593) = 5.2, p < 0.005] effects. Post-hoc testing revealed that there was an overall decrease in body weight for RK treatment compared with vehicle (p< 0.05). On days 26, 27, and 28, the RK group had a significantly reduced body weight compared with vehicle (p < 0.05 for all), see Fig. 2A. To further illustrate the treatment differences on high-fat induced weight gain, body weight was expressed as body weight change from baseline. For this, the pretreatment body weight was subtracted from daily body weight during treatment for 28 days. When expressed as body weight change, there were treatment effect [F (3, 59) = 5.7, p < 0.005], time effect [F (27, 1593) = 121.6, p < 0.005] and treatment × time [F (81, 1593) = 5.5, p < 0.005] effects. On Days 21-29, the RK mice weighed 2-2.5g less compared with the VEH group (p < 0.05 for all). On Days 26-29, the REH group weighed ~ 2 g less compared with the VEH group (p < 0.05 for all), see Fig. 2B. In one cohort, an OGTT was conducted after 3 weeks of dosing (n = 8 for REL, REH and RK; n = 7 for VEH) there was a time effect [F (6, 162) = 109.2, p< 0.005] and treatment × time effect [F (18, 162) = 2.1, p< 0.01]. At 15 min, both REH and RK had lower blood glucose levels compared with the VEH group (p < 0.05 for both), see Fig. 2 C. There were no differences among the groups for iAUC, see inset for Fig 2C. At the completion of the study, regional white adipose tissue was dissected from the carcasses. There was a significant effect for retroperitoneal fat [F (3, 55) = 4.8, p < 0.005], epididymal fat [F (3, 55) = 10.6, p < 0.005], and inguinal fat pads [F (3, 55) = 6.8, p < 0.005]. Post-hoc testing revealed that the REH and RK groups had lower fat pads mass than the VEH group for the retroperitoneal fat (p < 0.05 for both groups), inguinal fat (p < 0.05 for both groups), and the epididymal fat pads (p < 0.005 for both groups). In addition, REH and RK groups had lower fat pads mass than the REL group for retroperitoneal (p < 0.05 for both groups), inguinal fat (p < 0.05 for both groups), and the epidydimal fat pads (p < 0.005 for both groups), see Fig. 2D.

Fig. 2.

Daily treatment with phenolic-enriched raspberry extract on body weight, oral glucose, and white adipose tissue. Oral dosing treatments were vehicle (VEH; 50% propylene glycol, 40% water, and 10% DMSO), raspberry extract low (REL; 0.2 g/kg), raspberry extract high (REH; 2 g/kg), and raspberry ketone (0.2 g/kg). Data are represented as means ± SEM. A: Body weights for 28 days. Male C57BL/6J mice (n = 16 for REL, REH, and RK, n =15 for VEH) received oral dosing for 28 days. Data were analyzed with a one-way ANOVA with repeated measures. B: Weight change during treatment for 28 days. The pretreatment or baseline body weight was subtracted from daily body weight during treatment for 28 days. Data were analyzed with a one-way ANOVA with repeated measures. C: After 3 weeks of daily treatment, an OGTT was performed (n = 8 for REL, REH, and RK; n = 7 for VEH), inset shows incremental area under the curve (iAUC) of blood glucose following oral dosing. Data were analyzed with a one-way ANOVA with repeated measures. D: Carcass fat pad dissected after 28 days of treatment. Data were analyzed with an individual one-way ANOVA for each region. For each analysis, Newman-Keuls post-hoc testing was performed when justified, * indicates p < 0.05 from VEH, @ indicates REH is p < 0.05 from VEH, # indicates p < 0.05 from REL, ** indicates p < 0.005 from VEH, and ## indicates p < 0.005 from REL.

3.5. Cumulative food intake and meal patterns during the phenolic-enriched raspberry extract daily dosing regimen

There were no significant differences in cumulative food intake measured over the 28 days among the groups. Cumulative food intakes (g ± SEM) were 59.3 ± 0.3 for VEH, 60.8 ± 1.8 for REL, 60.6 ± 2.2 for REH, and 59.4 ± 0.4 for RK. To determine whether the body weight reduction and reduced adipose changes were a consequence of a reduction of feeding behaviors, meal patterns were measured in a separate cohort of mice during week 3 of the dosing protocols. Meal patterns were recorded over 3 days. There were no differences among groups for any parameter measures (Table 2).

Table 2.

Daily meal patterns of high-fat diet (45% fat) during phenolic-enriched raspberry extract treatments.

Meal Pattern Parameters	Vehicle	Raspberry Extract (Low; 0.2 g/kg)	Raspberry Extract (High; 2.0 g/kg)	Raspberry Ketone (0.2 g/kg)
Bouts	95.2 ± 2.9	78.1 ± 4.7	89.9 ± 6.8	77.2 ± 6.0
Meal Frequency	18.8 ± 0.6	15.6 ± 0.9	15.6 ± 1.0	16.3 ± 1.0
Average Meal Size (mg/meal)	105.8 ± 3.7	132.5 ± 7.5	133.8 ± 9.5	130.4 ± 10.4
Meal Duration (sec/meal)	536.7 ± 33.8	471.5 ± 23.4	557.5 ± 43.1	500.0 ± 51.7
Meal Rate (mg/min)	13.0 ± 1.0	17.9 ± 1.5	16.5 ± 1.8	19.6 ± 2.2
Daily meal Intake (g)	1.96 ± 0.06	1.95 ± 0.07	1.96 ± 0.13	1.98 ± 0.13

Meal patterns were measured over 3 days during the 3rd week of treatment in male C57BL/6J mice (n = 8 per group). Data are expressed as means ± SEM per 24-h period. Data were analyzed by a one-way ANOVA for each parameter.

3.6. Ambulatory activity following 4 weeks of daily dosing with phenolic-enriched raspberry extract.

Mice were dosed daily for 3 weeks prior to be housed in the indirect calorimetry system. Mice continued their respective dosing schedule and were habituated to the indirect calorimetry system for 6 days. Ambulatory activity and respiratory exchanges (see section 3.7) were measured on day 7 (day 28 of dosing). Ambulatory activity was binned into 1 h epochs over 24 h and is illustrated in Fig. 3A. There was an effect of total activity [F (3, 28) = 7.3, p < 0.001]. Post-hoc testing revealed that the REH group had the highest total activity compared with all groups (p < 0.01 for all), see Fig. 3B.

Fig 3.

Ambulatory activity (24 h) on Day 28 of daily treatments with phenolic-enriched raspberry extract. Oral dosing treatments were vehicle (VEH; n = 8), raspberry extract low dose (REL; 0.2 g/kg; n = 8), raspberry extract high dose (REH; 2 g/kg; n = 8), and raspberry ketone (0.2 g/kg; n=8). Male C57BL/6J mice were dosed at 1000 h. Data are represented as means ± SEM. Ambulatory activity was measured after 6 days of habituation. A: Hourly ambulatory activity over 24 h, shading indicates dark period, data were analyzed with a one-way ANOVA with repeated measures. B: Total ambulatory activity (total photo cell beam break counts) per group for 24 h. Data were analyzed with a one-way ANOVA. For each analysis, Newman-Keuls post-hoc testing was performed when justified, $ $ indicates p< 0.005 from all other groups.

3.7. Respiratory exchange and body composition following 4 weeks of daily dosing with phenolic-enriched raspberry extract.

EchoMRI measurements on day 29 demonstrated a difference in body composition. There was an effect for lean mass [F (3, 59) = 3.15, p < 0.05]. Post-hoc tests also revealed REL and RK groups had lower lean mass compared with VEH group (p < 0.05). There was also an effect for fat mass [F (3, 59) = 5.1, p< 0.005] and post-hoc testing revealed the REH and RK groups had lower fat mass compared with VEH group (p < 0.05), see Fig. 4A. RER and energy expenditure was estimated using indirect calorimetry on day 28 of dosing. There was a group effect on RER [F (3, 28) = 8.9, p < 0.005]. Post-hoc testing revealed that REL, REH, and RK had lower RER values compared with VEH group (p < 0.01), see Fig. 4B. Since lean body mass can influence body metabolism [34], energy expenditure was normalized to lean body mass. There was an effect for energy expenditure [F (3,28) = 3.1, p < 0.05]. Post-hoc testing revealed that REL had lower values compared with REH (p < 0.05), see Fig. 4C.

Fig 4.

Body composition and respiratory exchange (24 h) after 4 weeks of daily treatment with phenolic-enriched raspberry extract and raspberry ketone. Oral dosing treatments were vehicle (VEH; n = 8), raspberry extract low dose (REL; 0.2 g/kg; n = 8), raspberry extract high dose (REH; 2 g/kg; n = 8) and raspberry ketone (0.2 g/kg; n=8). Male C57BL/6J mice were dosed at 1000 h. Respiratory activity was measured by indirect calorimetry in the Comprehensive Lab Animal Monitoring System (Columbus Instruments) after 6 days of habituation. Represented respiratory measurements were on Day 28 of treatment. Body composition was measured on the following day using EchoMRI. Data are represented as means ± SEM. A: Fat and lean mass (in grams). B: RER (VCO₂/VO₂). C: Energy Expenditure (EE)/lean mass. Data were analyzed with a one-way ANOVA. For each analysis, Newman-Keuls post-hoc testing was performed when justified, * indicates p < 0.05 from vehicle; # indicates p < 0.05 from REL, $ indicates p < 0.05 from all other groups.

3.8. Terminal blood glucose, metabolic-related hormones, and HOMA-IR.

At the completion of the 28-day dosing protocol, mice (n = 15 for VEH, REL & REH n = 16, RK n = 16) were sacrificed in week 4 of treatment following a 5 h food deprivation. Trunk blood was assessed for terminal hormones and glucose. For active ghrelin, there was an effect [F (3, 57) = 3.5, p < 0.05], and post-hoc testing revealed that the REL, REH, RK had reductions from the VEH group (p < 0.05 for both), see Fig. 5A. There was also an effect for plasma insulin [F (3, 57) = 4.7, p < 0.01]. The REL had the highest insulin levels compared with all other groups (p < 0.05 for all), see Fig. 5B. The values of HOMA-IR were 29.98 ± 3.29 for VEH, 43.60 ± 4.68 for REL, 24.91 ± 3.33 for REH, and 25.97 ± 3.33 for RK. There was an effect for HOMA-IR [F (3,56) = 5.2, p<0.01] and post-hoc testing revealed REL had a higher score than other treatment groups. There were no significant differences in leptin and adiponectin levels, see Fig. 5C and Fig. 5D, respectively. For corticosterone, however, there was an effect [F (3,57) = 4.4, p < 0.01], post-hoc testing indicated REL corticosterone levels were higher than VEH (p < 0.005), see Fig. 5E. There were no differences in blood glucose values (in mg/dL ± SEM): 226 ± 13 for VEH, 231 ± 11 for REL, 221 ± 14 for REH, and 226 ± 11 for RK.

Fig 5.

Plasma hormone levels after 4 weeks of daily treatment with phenolic-enriched raspberry extract or raspberry ketone. Oral dosing treatments were vehicle (VEH; n = 15), raspberry extract low dose (REL; 0.2 g/kg; n = 16), raspberry extract high dose (REH; 2 g/kg; n = 16), and raspberry ketone (0.2 g/kg; n=16) in male C57BL/6J mice. Hormones were measured after a 5-h calorie deprivation. Data are represented as means ± SEM and were analyzed with an individual one-way ANOVA for each hormone. A: Active Ghrelin, B: Insulin, C: Leptin, D: Adiponectin, and E: Corticosterone. There were no differences in blood glucose values, not shown. For each analysis, Newman-Keuls post-hoc testing was performed when justified, * indicates p< 0.05 from VEH, ** indicates p < 0.005 from VEH, and $ indicates p< 0.05 from all other groups.

3.9. Effect on gene expression in the liver, ARC, and PVN

For Liver Hmox1 expression, there was an effect of treatment [F (3,56) = 20.9, p < 0.001]. There was an elevation in the REH group compared to all other groups (p < 0.001). For liver Pparg expression, there was an effect of treatment [F (3,56) = 3.9, p <0 .05]. REH group had reduced Pparg expression compared with RK and REL. For liver Lpl expression there was an effect of treatment [F (3,54) = 38.9, p < 0.001]. REH had increased expression compared with all other groups (p < 0.001), see Fig 6H. Liver Pgc1a did not have an overall difference between treatment groups. There was a trend towards increased expression of the critical transcription factor in the RK group (p = 0.053). However, there were no differences in the liver expression of Lipe, Ppara, Fas, Scd1. ARC and PVN gene markers of central control of food intake and energy homeostasis was assessed using qPCR. There were no differences in the ARC orexigenic gene markers Npy and Agrp, or the anorexigenic gene markers Pomc and Cart. Similarly, there were no treatment differences in the PVN Crh and Trh, or the adrenergic receptors Adra1a and Adra2c, see Fig 7.

Fig 6.

Hepatic markers of metabolism gene expression levels after 4 weeks of daily treatment with phenolic-enriched raspberry extract or raspberry ketone. Oral dosing treatments were vehicle (VEH), raspberry extract low dose (REL; 0.2 g/kg), raspberry extract high dose (REH; 2 g/kg) and raspberry ketone (0.2 g/kg) in male C57BL/6J mice. Data are represented as means ± SEM and were analyzed with an individual one-way ANOVA for each gene. A: Hmox1, B: Lipe, C: Ppara, D: Pparg, E: Pgc1a, F: Fas, G: Scd1, and H: Lp1. For each analysis, Newman-Keuls post-hoc testing was performed when justified. * indicates p< 0.05 from VEH, ** indicates p < 0.005 from VEH, $ indicates p< 0.05 from all other groups, and $$ indicates p< 0.005 from all other groups.

Fig 7.

Hypothalamic markers for feeding and metabolism gene expression after 4 weeks of daily treatment with phenolic-enriched raspberry extract or raspberry ketone. Hypothalamic tissue is from the arcuate (ARC) and paraventricular (PVN) nuclei. Oral dosing treatments were vehicle (VEH), raspberry extract low dose (REL; 0.2 g/kg), raspberry extract high dose (REH; 2 g/kg) and raspberry ketone (0.2 g/kg) in male C57BL/6J mice. Data are represented as means ± SEM and were analyzed with an individual one-way ANOVA for each gene. A: Npy, B: Pomc, C: Cart, D: Agrp, E: Adrala, F: Adra2c, G: Crh, and H: Trh.

4. Discussion

The present study investigated the preventative actions of a phenolic-enriched raspberry extract on the development of diet-induced weight gain and metabolic alterations in mice on a high-fat diet for four weeks. These preventative actions were compared with raspberry ketone. Based on our data, we accept our hypothesis that the phenolic-enriched raspberry extract promoted resilience to the metabolic alterations caused by an obesogenic diet. Our experimental approach was to daily administer by oral gavage either a low or high concentrations of a phenolic-enriched raspberry extract (0.2 g/kg or 2 g/kg, respectively). The outcomes of these treatments were compared with daily oral dosing of raspberry ketone (0.2 g/kg). To examine the preventative effects of these treatments on diet-induced weight gain, all dosing was initiated in mice coincident with the availability of high-fat diet (45% kcal fat). Specifically, we observed a reduction in weight gain with the high concentrations of phenolic-enriched raspberry extract and raspberry ketone. Previous studies with raspberry juice concentrate or raspberry puree (either 2.5% or 10% daily Kcal) supplemented to (i.e., added to the content of the diet) a high-fat diet (45% fat) and fed for 10 weeks have demonstrated a similar ability to prevent weight gain induced by a high-fat diet [5, 35]. The reduced weight gain observed with raspberry concentrates supplemented to the high-fat diet resulted in less hepatic lipid accumulation, but there were no differences in the inguinal fat mass [5, 35]. In the present study, we observed body weight reductions that were accompanied by a reduction in white adipose tissue. Reductions were observed in the retroperitoneal, epididymal, and inguinal fat mass depots. Our study also revealed a shift in body composition, in that based on body weight there was a decrease in the percentage of fat mass and an increase in the percentage of lean mass. Previously raspberry supplementation (5%) to a high-fat diet (60% fat) has been shown to reduce body weight gain, reduce inguinal and epididymal fat weights, and promote brown and beige adipose tissue formation after 10 weeks [8, 36].

Previous investigations have demonstrated that the weight loss and metabolic effects of the 5% raspberry supplementation were blocked in a conditional knockout of AMPKα1, suggesting that the obesity preventing effects of raspberry supplementation are dependent on AMPK-mediated pathways [8, 36]. Notably, significant differences in body weight with the 5% raspberry supplement were observed at 6 weeks [8, 36], whereas in our study differences in body weight gain were observed after 4 weeks with a high dose of the phenolic-enriched extract. Despite the difference in fat content of the diets (45% vs. 60%), the phenolic-enriched raspberry extract used in our study had a GAE total phenolic content of 37.6%, compared with ~1.1% GAE in the raspberry supplementation diet [8, 36]. This suggests that phenolic-enriched raspberry extract compared to the 5% raspberry supplementation diet has a similar preventative potential to reduce adiposity gain in response to a high-fat diet.

To ensure consistency and reproducibility of our studies, we used a commercially available whole-fruit raspberry juice concentrate as the starting material for our standard-prepared phenolic-enriched raspberry extracts. One limitation of this standard preparation was that the phenolic-enriched raspberry extract contained no detectable levels of raspberry ketone. In raspberries, raspberry ketone is synthesized from malonyl-CoA and p-coumaryl-CoA [10, 11]. The highest and detectable levels of intermediate and synthase/reductase enzymes are found in slightly overripe and somewhat dehydrated (i.e., stage 8) raspberries [10]. Because of the off-flavors associated with overripening, the raspberries used for whole-fruit concentrate were likely harvested at an earlier stage. The whole-fruit juice concentrate starting material was very low in dietary fibers (0 g/oz) but had a relative high sugar content (16 g/oz). To determine whether the remaining sugar content after the phenolic-enrichment process was sufficient to influence glucose homeostasis, we performed a tolerance test. Neither the phenolic-enriched extracts nor raspberry ketone had a differential action on blood glucose levels. Blood glucose was elevated when mice were acutely pretreated with high concentration of the phenolic-enriched raspberry extract 1 hour prior to an OGTT. Because there was an improvement of glucose tolerance in the OGTT at 30 min after 3 weeks of dosing with the high concentration of the phenolic-enriched raspberry extract, the elevation in glucose tolerance observed in the acute pretreatment OGTT could be related to the ability of the phenolic-enriched raspberry extract to facilitate glucose absorption. One limitation of our study, however, was that we did not include a group of mice in our chronic dosing experimental design that were fed a low-fat diet or standard chow. Therefore, it is possible that the repeated dosing with phenolic-enriched raspberry extract impairs glucose tolerance under normal feeding conditions (i.e., non-obesogenic). The influence of the phenolic-enriched extract on blood glucose homeostasis will be examined in future studies. Notably, in our study, raspberry ketone (0.2 g/kg) did not alter blood glucose values in the acute tolerance test or in the pretreatment with the OGTT, but we observed this dose of raspberry ketone was effective at preventing body weight and white adipose tissue mass in mice fed a high-fat diet. Based on structural-activity relationship, raspberry ketone has been suggested to have actions that inhibit α-glucosidase [37]. Our results suggest that an acute dose of raspberry ketone does not influence blood glucose levels either alone or after oral dosing with the monosaccharide, glucose, in vivo.

Another finding from our study was that plasma ghrelin, a gut peptide directly associated with subjective feelings of hunger in humans and with food-seeking behavior in animals[38], was reduced in the phenolic-enriched raspberry extract- and raspberry ketone-treated groups. Despite this finding, we did not observe a reduction in cumulative calorie intake or alterations in the meal patterns with the phenolic-enriched raspberry extract compared with the vehicle-treated group. A previous study by Cotten and colleagues fed mice a high-fat diet (45% fat kcal) containing raspberry ketone (0.25% or 1.74%) for 5 weeks and observed a reduction in cumulative calorie intake[20]. Mice fed the raspberry ketone supplemented diet also had reduced white adipose mass, but the reduction in white adipose tissue were similar to a group calorie matched to the raspberry ketone-fed mice (0.25%) (i.e., pair-fed). As a result, Cotten and colleagues concluded that the ability of raspberry ketone to reduce adiposity was a consequence of reduced energy intake[20]. Although our findings are similar to those of Cotten and colleagues in that we did not observe differences in plasma adiponectin levels [20], our findings suggest that preventative actions of phenolic-enriched raspberry extract and raspberry ketone on weight gain are a consequence of the potential to reduce white adipose tissue accumulation, not secondary to a reduction in energy intake. The discrepancy between our findings and those of Cotten and colleagues is probably due to the dosing differences in how the raspberry ketone was administered, which likely influenced the bioaccessibility and bioavailability of raspberry ketone. Another concern regarding the supplementation of raspberry ketone or phenolic compounds in the diets is how diet supplementation affects palatability and hardness of the pelleted diet. Diet palatability was not tested in previous investigations with raspberry ketone or raspberry supplementation [8, 20, 36]. Ongoing studies from our lab are investigating the most effective methods for increasing the bioavailability of raspberry-derived phenolics and related bioactive materials.

Another finding from our study was that phenolic-enriched raspberry extract and raspberry ketone reduced the respiratory exchange ratio (RER), which indicates greater fat oxidation, after 4 weeks of daily dosing. All treatments indicated lower RER from vehicle treatment. However, the low (0.2g/kg) concentration of the phenolic-enriched raspberry extract was only significantly lower from the high (2g/kg) concentration of the phenolic-enriched raspberry extract when energy expenditure was normalized to lean mass. In addition, the low concentration of the phenolic-enriched raspberry extract resulted in elevated plasma insulin, HOMA-IR, and corticosterone. Taken together, this suggest that low concentration of the phenolic-enriched raspberry extract could be reducing energy expenditure through adverse effects. This possibility will be examined in future studies.

We found an increased expression of Hmox1 and Lpl in the hepatic tissue of REH treated mice. Increased levels of Lpl may play a role in improved triglyceride and total cholesterol clearance from the circulation and can help ameliorate the effects of a high-fat diet in the REH treated animals [39]. One limitation of our study, however, was that we did measure plasma and hepatic lipids levels. Raspberry ketone has been shown to dose-dependently increase Hmox1 to reduce adipose size and inhibit adipogenesis signaling in vitro [15, 21]. Hmox1 is an inducible, rate-limiting cellular enzyme that cleaves the α-mesocarbon bridge of heme and heme-related structures to generate CO, iron ions, and bile pigments [40]. The role of Hmox1 in vivo, however, remains unclear. For instance, orally administered raspberry ketone (160 mg/kg) for 8 weeks prevented ovariectomized weight gain and restricted adipocyte size, but the inguinal fat expression of Hmox1 was not different in the ovariectomized mice and ovariectomized mice treated with raspberry ketone [15]. Interestingly, mice fed a high-fat diet adulterated with raspberry puree (2.5% kcal) for 10 weeks had increased hepatic expression of Hmox1 and reduced hepatic lipid content [35]. In our study, the REH animals had an increased expression of Hmox1 which can may play a role in antioxidant mechanisms. Interestingly the RK treated animals did not show increased Hmox1 expression. This further suggests a polyphenol enriched raspberry extract and raspberry ketone affect different metabolic pathways. The chemical profile of the phenolic-enriched raspberry extract revealed that predominant anthocyanins were glycosylated cyanidins, whereas the predominant ellagitannin was sanguiin-H6. One speculation is that combination of these two bioactive phytochemicals could be responsible for the differential effects of phenolic-enriched raspberry extract compared with raspberry ketone. Future studies will examine the combinational interactions of these phytochemicals on weight gain prevention in an obesogenic environment.

Raspberry ketone and raspberry derived polyphenols have been noted to reduce markers of inflammation [9, 41–43]. Chronic low-grade inflammation and cytokine signaling promote diet -induced metabolic alterations associated with excessive weight gain [44]. Oral dosing of raspberry ketone in rats for 4 weeks has been shown to reduce the cytokine profile associated with diet-induced steatohepatitis and isoproterenol-induced cardiac damage [41,42]. Mice fed a high-fat diet (45% fat) containing raspberry-derived anthocyanins (200 mg/kg) demonstrated a reduction in weight gain after 12 weeks, which was accompanied by an improved profile of hepatic cytokines and markers of oxidative stress [43]. More extensive cytokine profiling and antioxidant actions of the phenolic-enriched raspberry extracts in mice with a more protracted (> 4 weeks) high-fat diet exposure are needed.

In conclusion, our data demonstrated that a standard-prepared phenolic-enriched raspberry extract (2 g/kg) reduces adiposity gain following consumption of a high-fat diet for 4 weeks. Even though the preventative actions on weight gain were more pronounced with raspberry ketone (200 mg/kg), phenolic-enriched treatments resulted in decreased ghrelin levels, hepatic metabolic markers, and alterations in respiratory exchanges. Future studies will characterize the metabolic signature of a standard - prepared phenolic-enriched raspberry extract and raspberry ketone to further understand the preventative actions on the development of diet-induced obesity.

Abbreviations:

Adra1a

Adrenoceptor alpha 1a

Adra2c

Adrenoceptor alpha 2c

Agrp

Agouti related peptide

AMPK

AMP-activated protein kinase

ANOVA

Analysis of Variance

ARC

Arcuate nucleus

AUC

Area under the curve

BioDAQ

Biological Data Acquisition System

Cart

Cocaine and amphetamine regulated transcript

Crh

Corticotropin releasing hormone

DAD

Diode Array Detector

DEPC

Diethylpyrocarbonate

DMSO

Dimethyl sulfoxide

EE

Energy expenditure

ESI

Electrospray ionization

EtOAc

Ethyl acetate

Fas

Fatty acid synthase

GAE

Gallic acid equivalent

Gapdh

Glyceraldehyde-3-phosphate dehydrogenase

Hmox1

Heme oxygenase 1

Hprt

Hypoxanthin-guanine phosphoribosyltransferase

LOD

Limit of detection

Npy

Neuropeptide Y

OGTT

Oral glucose tolerance test

Pgc1a

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

Ppara

Peroxisome proliferator-activated receptor alpha

Pparg

Peroxisome proliferator-activated receptor gamma

PVN

Paraventricular nucleus

REH

Raspberry extract high (2 g/kg)

REL

Raspberry extract low (0.2 g/kg)

RER

Respiratory Exchange Ratio

RK

Raspberry ketone (0.2 g/kg)

Scd1

Stearoyl-CoA desaturase

SEM

Standard error of mean

TFA

Trifluoroacetic acid

Trh

Thyrotropin releasing hormone

VEH

Vehicle