Journal of Food and Nutritional DisordersISSN: 2324-9323

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Research Article, J Food Nutr Disor Vol: 3 Issue: 4

Blackberry (Rubus sp. var. Loch Ness) Extract Reduces Obesity Induced by a Cafeteria Diet and Affects the Lipophilic Metabolomic Profile in Rats

Kenia Bispo1, Marcel Piovezan2, Daniel García-Seco3, Encarnación Amusquivar3, Danuta Dudzik3, Beatriz Ramos-Solano3, Javier Gutiérrez-Mañero3, Coral Barbas3 and Emilio Herrera4*
1CAPES Foundation, Ministry of Education of Brazil, Brasília – DF 70040-020, Brazil
2Department of Chemistry, Federal University of Santa Catarina, Florianopolis, SC, Brazil
3Faculty of Pharmacy, Universidad San Pablo CEU, Madrid, Spain
4Professor of Biochemistry and Molecular Biology, University CEU San Pablo, Madrid, Spain
Corresponding author : Herrera E
Professor of Biochemistry and Molecular Biology, University CEU San Pablo, Madrid, Spain, Universidad San Pablo CEU, Ctra. Boadilla del Monte km 5.300, 28668-Boadilla del Monte (Madrid), Spain
Tel: +34 913724730; Fax: +34 913510496
E-mail: eherrera@ceu.es
Received: April 9, 2014 Accepted: July 18, 2014 Published: July 22, 2014
Citation: Bispo K, Piovezan M, García-Seco D, Amusquivar E, Dudzik D, et al. (2014) Blackberry (Rubus sp. var. Loch Ness) Extract Reduces Obesity Induced by a Cafeteria Diet and Affects the Lipophilic Metabolomic Profile in Rats. J Food Nutr Disor 3:4. doi:10.4172/2324-9323.1000149

Abstract

Blackberry (Rubus sp. var. Loch Ness) Extract Reduces Obesity Induced by a Cafeteria Diet and Affects the Lipophilic Metabolomic Profile in Rats

Blackberries (Rubus sp. var. Loch Ness) contain large amounts of anthocyanins and flavonols, which have several health benefits. The present study was designed to determine the effects of a methanolic blackberry extract in rats fed a cafeteria diet. Weaned female rats were assigned to one of three dietary groups: standard pellet diet (SD), cafeteria diet (CD) and cafeteria diet supplemented with Rubus extract (CRD) for 90 days. Plasma metabolites and insulin were analyzed with commercial kits and fatty acid profile was measured by gas chromatography whereas other aliquots were subjected to metabolomics fingerprinting analysis using ultra high efficiency liquid chromatography. Lipoprotein lipase (LPL) activity was determined in fat depots by a radiochemical method. In comparison to the SD group, rats of the CD and CRD groups had increased plasma myristic, palmitic and oleic acids and those of the CD group had increased liver and different adipose tissue weights; t .

Keywords: Blackberry fruit extract; Adipose tissue; Insulin sensitivity; Lipoprotein lipase; Metabolomic profile; Rats

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Keywords
Blackberry fruit extract; Adipose tissue; Insulin sensitivity; Lipoprotein lipase; Metabolomic profile; Rats

Introduction
Anthocyanins have several health benefits such as preventing cholesterol-induced atherosclerosis [1], inhibiting platelet aggregation [2] and having antiinflammatory [3] and anticarcinogenic [4] activities. Blackberry has a high content of phenolic compounds, which have been shown to inhibit oxidation of human LDL and lecithin liposomes [5] in vitro. Furthermore, blackberry anthocyanins suppress cancer cell growth by modifying cancer cell signaling pathways [6,7] and have been shown to improve body weight and body composition and to reduce obesity in mice [8]. Nevertheless, the health benefits of blackberry have not been sufficiently explored [9].
Rodents fed a high-fat diet rapidly develop insulin resistance and impaired activation of the insulin-signaling pathway [10-13]. However, high-fat feeding has been considered a radical dietary intervention, whereas a cafeteria diet, which is a highly palatable hypercaloric diet with a more balanced caloric composition, better resembles a Western diet [14]. The cafeteria diet – composed of highfat and high-sugar supermarket products – results in obesity, glucose intolerance and insulin resistance in rats [15,16] and hamsters [17], and reduced insulin clearance in mice [18], and has been considered a robust model of metabolic syndrome in humans [19].
Studies carried out in animal models generally focus on a series of metabolic markers or parameters, previously defined as the best or most studied indicators of a given disease. However, little is known about other metabolites that are not considered in these studies. The development of metabolomic techniques is enabling these gaps in knowledge to be corrected; the huge amount of data provided by these tools combined with multivariate analysis can reveal those factors (metabolites) where the diseased condition differs from the normal (healthy) condition [20]. The benefits of a holistic approach using metabolomics may result in identification of new metabolic markers to predict the development of some diseases [21] or in drawing unexpected conclusions when correlations appear.
On the basis of the small amount of information available about the metabolic effects of blackberry, we aimed to determine the effects of a blackberry extract on some metabolic variables and on the lipophilic metabolomics profile in rats fed a cafeteria diet. In order to determine the actual response of the rats to the cafeteria diet, another group of animals fed the standard diet was studied in parallel. Since different sex responses to both high-fat diet [22] and cafeteria diet [23] in terms of adiposity and lipid handling have previously been reported, the present study was carried out exclusively in adult female rats. Multivariate analysis of lipophilic metabolomic profiles was used to integrate all the data.
The results show the expected increase in fat depots and in adipose tissue lipoprotein lipase activity, as well as the low insulin sensitivity index and changes in the metabolomics profile in the rats fed the cafeteria diet. They also show that the dietary supplement with blackberry extract can reduce the impact of the cafeteria diet on all these variables.

Materials and Methods
Preparation of the Rubus extract
Blackberries (Rubus sp. var. Lochness) were kindly provided by Agricola El Bosque (Lucena del Puerto, Huelva, Spain). Rubus extracts were obtained by lyophilization and extraction with 80% methanol in water as previously described [24]. For determination of total anthocyanin, the extract was diluted 1:9 (v/v) in methanol, and anthocyanin content was determined quantitatively by the pH differential method previously described [25] with minor modifications. The concentration of anthocyanins was 5.42 g of cyanidin-3-glucoside per 100 g of Rubus extract.
Flavonoid content was measured by the aluminum chloride assay [26] using catechin (Sigma Chemical Co., St. Louis, MO) as standard; (-)epicatechin was the predominant flavonoid as described in [27] with a content of 499 ± 8 mg of epicatechin per 100 g of Rubus extract.
Animals and experimental design
Female Sprague Dawley rats were obtained from the animal quarter of University San Pablo CEU, Madrid, Spain. The experimental protocol was approved by the Animal Research Committee of the University San Pablo CEU. The rats were weaned at 21 days of age, placed in collective cages (5 per cage) under controlled conditions (22 ± 2°C, 55 ± 10% relative humidity and constant cycle light/dark of 12 h with continuous ventilation). Rats were given a standard pellet diet (Harlan Global Diet 2014, Madison, WI) for 5 days, after which they were randomly assigned to one of three dietary treatment groups: the standard diet group (SD) was maintained on the pellet diet, the cafeteria diet group (CD) was given the cafeteria diet, and the cafeteria plus Rubus diet group (CRD) was given the cafeteria diet supplemented with the Rubus extract. The cafeteria diets were based in those previously reported by others [28-30] and prepared as a homogeneous paste by mechanically (Sammic, Guipúzcoa, Spain) blending the components. Composition and fatty acid profile of the diets are shown in Table 1 and 2 respectively.
Table 1: Composition of the diets per 100 g
Table 2: Concentration of fatty acids in the experimental diets (mg/g)
The CD and CRD were stored at -20ºC until use. Rats had free access to the assigned diet and tap water. After 80 days on the experimental diets, rats were subjected to an oral glucose tolerance test (OGTT) that was performed as follows. Tests were conducted between 11:00 and 13:00 after a 3 h fast. After tail blood was collected (time 0), rats received an oral load of 2 g glucose/kg body weight, and blood was collected at 7.5, 15, 30 and 60 min into tubes containing 1 g Na2EDTA/L. Plasma was separated by centrifugation at 1,500 g for 15 min at 4ºC and stored at -80ºC until analyzed for glucose and insulin. The insulin sensitivity index (ISI) was calculated as previously described [31] using the following equation: ISI = 10,000/√(FPG x FPI x mean G x mean I) where FPG is fasting plasma glucose (in mg/dL), FPI is fasting plasma insulin (in μL/mL) , and mean G and mean I are the mean glucose and mean insulin concentrations in the same units determined during the OGTT. One week after the OGTT, rats were sacrificed using a guillotine while under CO2 anesthesia and trunk blood collected into ice-chilled tubes containing 1 g Na2EDTA/L. Plasma was separated from fresh blood and stored as described above. Liver and different fat depots were rapidly dissected and placed into liquid nitrogen for weighing, and fat depots were stored at -80ºC until analysis.
Processing of the metabolic variables
Plasma glucose, triacylglycerols (TAG) and cholesterol (Spinreact Reactives, Spain) and non-esterified fatty acids (NEFA) (Wako Chemicals, Germany) were determined with commercial kits by enzymatic methods and insulin was analyzed by ELISA (Mercodia, Sweden). For the analysis of the fatty acids profile, nonadecenoic acid (19:1) (Sigma Chemical Co.) was added as the internal standard to fresh aliquots of each diet and of frozen plasma, which were used for lipid extraction and purification [32]. The final lipid extract was evaporated to dryness under vacuum and the residue resuspended in methanol/toluene and subjected to methanolysis in the presence of acetyl chloride at 80ºC for 2.5 h as previously described [33]. Fatty acid methyl esters were separated and quantified on a Perkin-Elmer gas chromatograph (Autosystem) with a flame ionization detector and a 20 m Omegawax capillary column (internal diameter 0.25 mm). Nitrogen was used as carrier gas, and the fatty acid methyl esters were compared with purified standards (Sigma Chemical Co.). Quantification of the fatty acids in the sample was performed as a function of the corresponding peak areas compared to that of the internal standard. Lipoprotein lipase (LPL) activity was assayed in inguinal and lumbar fat depots in acetone/diethyl ether extracts by the conversion of triolein, [carboxyl-14C] (Perkin Elmer, Boston, MA) to [1-14C]-oleic acid as previously described [34].
Metabolomic analysis
LC-MS grade organic solvents and reagents for the metabolomics analysis were purchased from Fluka Analytical (Sigma – AldrichChemie GmbH, Steinheim, Germany).
Plasma samples were thawed in ice. To remove proteins from the samples, 3 volumes of ice-cold methanol/ethanol 1:1 (v/v) were added to each plasma aliquot and incubated in ice for 5 min. After centrifugation at 16,000 rpm and 4°C for 20 min, supernatants were filtered through a 0.22 μm nylon filter. Quality control (QC) was determined [35] in samples that were prepared independently by following the same protocol by pooling equal volumes from each plasma sample.
The metabolomic fingerprinting analysis of plasma was performed using ultra-high efficiency liquid chromatography (UHPLC) (Agilent 1290 Infinity LC System) in 0.5 μL of extracted plasma samples that were injected to a reverse-phase Zorbax Extend C18 column (2.1 × 50 mm, 1.8 μm, Agilent Technologies) at 60ºC. The composition of the mobile phases was: A - water with 0.1% (v/v) formic acid, and B - acetonitrile with 0.1% (v/v) formic acid. The chromatographic gradient using a constant flow rate of 0.6 mL/min was started at 5% phase B for the first minute, increasing to 80% from 1-7 min, then to 100% from 7-11.5 min, holding at 100% for 0.5 min, finally returning to 5% of phase B from 12 until 15 min (system re-equilibration). Samples were analyzed in positive ESI(+) and negative ESI(-) ionization modes in separate runs of MS, MS/MS analysis, respectively operated in full scan mode from 50-1000 m/z for positive and 50-1100 m/z for negative mode. Capillary voltage was set to 3 kV for positive and negative ionization mode; fragmentor voltage was set to 175 V for positive and 250 V for negative ionization mode; the drying gas flow rate was 12 L/min at 250ºC and gas nebulizer 52 psi. Samples were injected in randomized order in two runs (for positive and negative ion mode). At the beginning of each run, a batch of 10 injections of QC samples was used to condition the column.
For the metabolomics study, MassHunter Workstation Software LC/MS Data Acquisition version B.05.00 (Agilent Technologies) was used for control, acquisition and processing of all data obtained with UHPLC–QTOF/MS. The resulting data file was cleaned of background noise and unrelated ions by the Molecular Feature Extraction (MFE) tool. Alignment and data filtering were performed by Mass Profiler Professional (MPP, version B.12.1, Agilent Technologies) software. Accurate masses of features were searched for possible structure against the online databases such as CEU mass mediator (http:// ceumass.eps.uspceu.es/mediator), METLIN (http:// metlin.scripps.edu), HMDB (http://hmdb.ca), KEGG (http:// genome.jp/kegg) and LipidMaps (http://lipidmaps.org). The identity of compounds was confirmed by LC-MS/MS by using a QTOF (6550 system, Agilent Technologies) with the same chromatographic conditions as used in the primary analysis. Ions were targeted for collision-induced dissociation (CID) fragmentation on the fly, based on the previously determined accurate mass and retention time. Comparison of the structure of the proposed compound with the obtained fragments as well as comparison with the retention time and isotopic distribution of commercially available standards was used to yield to final confirmation of the identity of metabolites.
Statistics and data processing
Statistical analysis for the metabolic variables was carried out using GraphPad Prism 5.O. (GraphPad Software Inc. La Jolla, 115 CA). After checking a normal distribution of the data using the Kolmogorov- Smirnov test, one-way analysis of variance (ANOVA) was used to compare different diets. Bartlett’s test was used to prove homogeneity of the variance. When treatment effects were significantly different (p <0.05), Newman-Keuls simultaneous tests were used to establish statistical differences between individual dietary interventions.
For data provided by the metabolomics study, normality was verified by evaluation of the Kolomgorow-Smirnov-Lillefors test and variance ratio by the Levene’s test. Differences between experimental groups were performed by one way ANOVA (equal or unequal variance) or non-parametric Kruskal-Wallis test. The levels of statistical significance were set at p<0.05. Statistical analysis were performed using Matlab R2010a (Mathworks) software. The multivariate analysis, statistical calculations and plottings were obtained with SIMCA P+ 12.0 (Umetrics, Umea, Sweden).d

Results
Metabolic changes
As shown in Figure 1a, daily food intake by rats of the three groups was similar throughout all the experiment, although due to the higher caloric content of the cafeteria diet the daily energy intake of rats in groups CD and CRD was higher than those in the SD group (Figure 1b).
Figure 1: Mean ± SEM of 5 rats/group for daily food (a) and energy intake (b) at different days of experiment in rats fed standard diet (SD), cafeteria diet (CD) or cafeteria plus Rubus extract diet (CRD). Asterisks indicate statistical significant difference of the CD or CRD groups versus the SD group whereas no difference was found between these two groups for energy intake nor between the three groups for food intake.
As well as having higher caloric content, cafeteria diets (i.e. both CD and CRD) contain more saturated and monounsaturated fatty acids than the standard diet (SD), whereas their content of n-6 polyunsaturated fatty acids (PUFA) mainly corresponding to linoleic acid (18:2 n-6), is similar and that of n-3 fatty acids mainly corresponding to α-linolenic acid (18-2 n-3), is slightly higher (Table 2). The different fatty acid composition of the diets affected the level of specific fatty acids in plasma: myristic (14:0), palmitic (16:0) and oleic (18:1, n-9) acids were higher in both the CD and CRD groups than in the SD group, whereas no significant differences were found in the plasma concentrations of either stearic acid (18:0) or any of the PUFA between groups (Table 3).
Table 3: Fatty acid concentration in plasma (mg/L) of female rats that were fed with standard diet (SD), cafeteria diet (CD) or cafeteria diet supplemented with Rubus extract (CRD) for 90 days, when they were sacrificed by decapitation after a 3 h fast.
Body, liver and different fat depot weights and plasma metabolic variables of the rats are shown in Table 4.
Table 4: Body, liver and different fat depots weights and plasma metabolic variables in female rats that were fed with standard diet (SD), cafeteria diet (CD) or cafeteria diet supplemented with Rubus extract (CRD) for 90 days, when they were sacrificed by decapitation after a 3 h fast.
The consumption of either CD or CRD did not increase body weight, whereas liver weight was higher in the CD group than in SD (p <0.05), the effect disappearing in the CRD group. All the adipose tissue depots studied (inguinal, perirenal, mesenteric and lumbar adipose tissue) showed a significantly higher weight in the rats on CD than in those on SD, and this effect appeared lower in those rats fed the CRD – in the case of inguinal and mesenteric adipose tissues, the difference compared to the SD was no longer significant (p >0.05). Basal plasma glucose and insulin levels did not differ between the groups, whereas the area under the curve (AUC) for both glucose and insulin after the oral glucose load (OGTT) was higher in rats fed the CD than the SD. The Rubus supplement (CRD) did not modify the augmented AUC for glucose seen in the CD rats but it decreased the AUC for insulin to values that were not significantly different from either of the other two groups. Values of plasma glucose and insulin, both basal and after the oral glucose load, were used to determine insulin sensitivity index (ISI). The ISI values calculated were lower in the two groups fed the CD than in those on SD, with no difference observed between those receiving or not receiving the Rubus supplement (Table 4). Plasma triacylglycerols were higher in those rats fed the CD or the CRD than on those on the SD although the difference was only significant in the case of the CRD group. However, neither NEFA nor cholesterol concentrations differed among the three groups.
LPL activity was measured in both inguinal and lumbar adipose tissue. As shown in Table 5, the LPL activity of inguinal adipose tissue was higher in rats on the CD group than in those on SD; once again this variable decreased in rats on the CRD group to values that no longer differed from the SD group. A similar trend was found in LPL activity of the lumbar adipose tissue, although the differences among the groups did not reach statistical significance due to the high standard error values.
Table 5: Lipoprotein lipase (LPL) activity (pkats/mg protein) in inguinal and lumbar adipose tissue of female rats that were fed standard diet (SD), cafeteria diet (CD) or cafeteria diet supplemented with Rubus extract (CRD) or 90 days, when they were sacrificed by decapitation after a 3 h fast.
Metabolomic variables
The common statistical approach used in metabolomics data analysis is based on univariate and multivariate analysis (MVA). To evaluate the quality of controls (QC), samples were first tested by unsupervised principal components analysis (PCA-X). QCs were clustering together (data not shown), reflecting the system’s stability and performance, and the repeatability of the sample treatment procedure [36]. The coefficient of variation (% CV) of QC samples was calculated and values are shown in Table 6.
Table 6: Most relevant metabolomics changes in plasma, related to specified groups of the rats that were fed with standard diet (SD), cafeteria diet (CD) or cafeteria died supplemented by Rubus extract.
In fact, the experimental variables can only be considered significantly different when the percentage of change between groups is higher than the %CV for the corresponding QC.
Figure 2 (panels A and C), shows a trend to SD group clustering independently from CD and CDR groups only in the negative MS polarity when a not-supervised principal components analysis (PCAX) of the results was employed.
Figure 2: Panel A unsupervised principal component analysis (PCA) for the data in electrospray inonization (ESI)(+) and Panel C for the data in ESI(-). Panel B Partial Least Squares-Discriminat Analysis (PLS-DA) plot of analyzed samples in ESI(+) and Panel D in ESI(-). Legend: Standard diet (SD) (), Cafeteria diet (CD) (), Cafeteria + Rubus diet (CRD) (). N= 5 rats/group.
In the supervised models, a better separation among the three groups (SD, CD and CDR) is seen (Figure 2, panels B and D), where the variance explained was R2=0.77 (+) ESI, R2=0, 85 () ESI and variance predicted Q2=0.14 (+) ESI, Q2=0.52 (-) ESI. Differences between the groups became more obvious when they were compared as pairs (CD vs. CRD, CD vs. SD and CRD vs. SD) as it is shown in Table 6.
When comparing rats of the CD group vs. those of SD (Table 6), it was found that oleic acid and most of the acylcarnitine derivatives, especially stearoylcarnitine (C18:0) were up-regulated in the CD group. Supplementation with Rubus extract to the CD group (CRD) provides a clear metabolic change compared to both CD and SD groups. Although, no significant changes between CD vs. CRD groups were found, a clear tendency to down-regulate acylcarnitine derivatives in the CRD group was observed. Interestingly, a remarkable impact of the Rubus supplement to cafeteria diet (CRD) as compared to the standard diet (CRD vs. SD) is evidenced, since the significant effect of cafeteria diet (CD) vs. SD decreases when the former is supplemented with the Rubus extract (CRD).
In general, most of the glycerophospholipids including phosphatidylcholines (PC), lysophosphatidylcholines (lysoPC) and lysophosphatidylethanolamines (lysoPE) were significantly downregulated in both CD and CRD vs. SD, without any significant differences between CD and CRD (Table 6). Some of the lysophosphatidylcholines like lysoPC(14:0) and lysoPC(17:0) with saturated fatty acid chain were up-regulated in the CD group as compared to SD, although the differences were not significant. However, when the same variables were compared between CRD and SD, the differences were statistically significant. It’s worth mentioning CerP, another compound that was elevated in CD in comparison to SD, the difference disappearing when comparing CRD and SD.

Discussion
In this study it was found that a hypercaloric diet containing a high proportion of saturated fatty acids to young female rats over 90 days increased the mass of fat depots and plasma triacylglycerol concentrations and decreased the insulin sensitivity index studied after an oral glucose load. Further, the plasma profile of fatty acids in these animals (again compared to standard diet controls) showed significant increments in saturated fatty acids without a change in the level of PUFA, and they had increased fat depots. These findings show that the dietary model of energy-dense palatable food applied here (i.e. the socalled cafeteria diet) to young female rats corresponds to a dietinduced obesity that mimics metabolic syndrome in humans, in agreement with a previous proposal [19]. The detected increase in adipose tissue LPL activity in the rats fed the CD is consistent with the increase in the sizes of fat depots, and indicates that their hypertriacylglycerolemia did not result from reduced clearance of circulating triacylglycerols by extrahepatic tissues, but more likely from an increased production by the liver. Treatment of animals fed the CD with an extract of Rubus (CRD) had a small effect on plasma lipid components, probably as a consequence of their low insulin sensitivity, which was not modified by this treatment. However, some consistent decline in liver and inguinal and mesenteric adipose tissue mass could be detected in rats fed the CRD. Such a change in adipose tissue mass is consistent with the decline in LPL activity found in these animals, but the mechanism involved will require additional investigation.
In order to understand further the metabolic changes caused by the treatments, the highly sensitive and reproducible LC-QTOF-MS tool for metabolomic analysis has been employed to study plasma aliquots. The multivariate analysis carried out considering all data from the lipophilic metabolic profiles demonstrated a clear separation of the three groups (Figure 2) and indicated which metabolites were responsible for the separation, with statistical significance (Table 6). The main findings from the metabolomic fingerprinting in the case of the cafeteria diet are related to phospholipids, mainly zwitterionic glycerphospholipids, the related lysophospholipids as well as longchain acylcarnitines. The carnitine ester profiles (tetradecanoylcarnitine 14:0; palmitoylcarnitine 16:0 and stearoylcarnitine 18:0) tended to be higher in the CD group than in the SD, however, linoleylcarnitine 18:2 is observed to be down-regulated. Acylcarnitines are ester derivatives of carnitine, the homeostasis of which is maintained by dietary intake, a modest rate of endogenous synthesis from lysine and methionine and by renal reabsorption. The carnitine system, including free carnitine and acylcarnitines, is essential for the transport of long-chain fatty acids from cytoplasm into mitochondria for their subsequent oxidation [37]. Interestingly, our findings fit to the one mass spectrometry based metabolic profiling reported by Koves, et al. [38] showing that long-chain acylcarnitines (16:0, 18:0 and 18:1) are increased in diet-induced obese rats. Higher levels of long-chain saturated and monounsaturated acylcarnitine species, analyzed by tandem mass spectrometry (MS/MS), in obese and insulin-resistant subjects compared to lean controls have also been shown in human obesity [39]. It may therefore be possible that, in the circumstances of our rats on the cafeteria diet, an inefficient tissue fatty acid beta-oxidation, due in part to a relatively low tricarboxylic acid cycle capacity, generates acylcarnitine molecules that activate proinflammatory pathways implicated in insulin resistance, as hypothesized for type 2 diabetic women [40].
A specific accumulation of saturated acylcarnitines was found in plasma of rats fed the cafeteria diet, indicating inefficient betaoxidation [41,42], which together with the abundance of these fatty acids in the diet would contribute to their higher concentrations in plasma.
The greatest difference in the metabolomic profiles found here among the three studied groups (i.e. SD, CD and CRD groups) was in the glycerophospholipids which biological properties are dependent to differences of their structure and fatty acids composition (chain length, position, degrees of saturation and double bond location). The main group of identified glycerophospholipids, as lysoPC with the shorter chain fatty acids (14:0, 17:0, 18:0) and lysoPE(P-16:0), lysoPE(20:1)), showed up-regulation in rats fed the CD vs SD, whereas those with the long chain fatty acids, mainly polyunsaturated, were down-regulated in these same rats (i.e. in the CD group). It appears that Rubus supplementation reduce these changes in rats fed with the CD diet, making their comparison with the SD much less significant.
In general, a reduction in plasma lysoPC species in the rats fed the CD was found (Table 6). Our data are similar to others’, which demonstrated that plasma lysoPC concentrations are reduced in mice fed a high fat diet [43] and in human obese subjects [44]. The mechanism responsible for the reduction in circulating lysoPC in these conditions is unknown, but could be related to an increase in either its breakdown or its clearance from the circulation by metabolically active tissues, as previously proposed for newly diagnosed type 2 diabetic subjects [45].
Furthermore, recently lysoPC has also been shown to have a role in the metabolism of glucose. It has been reported that lysoPC activates glucose uptake by adipocytes and that after acute lysoPC administration to mouse models of diabetes, there is an improvement in their glycemia [46]. In agreement with this involvement of plasma lysoPC in the glucose homeostasis, our rats fed the CD, with lower lysoPC levels than the SD group, showed a glucose metabolism that was somehow affected as revealed by their higher AUC for both insulin and glucose in the OGTT, and the lower ISI values as compared to those in rats fed the SD.
Knowledge about lysophosphatidylethanolamines (lysoPE) is not as wide but, it seems that they can be synthesized in a similar way to lysoPC [47]. In agreement with this view, our study shows that changes in lysoPE levels in CD and SD rats were similar, being upregulated as in case of lysoPE(P-16:0) and lysoPE(20:1) or downregulated as lysoPE(18:1). Some of the changes induced by the CD observed on the metabolomic parametres (Table 6) disappear after the Rubus supplement, for example, LysoPC18:0, LysoPC20:1, LysoPC22:4, LysoPC22:5 LysoPC24:0, LysoPA16:0, CerP18:1 CerP18:0. Although their physiological meaning is not well known, the changes in these compounds appear as putative indicators of the metabolic alteration caused by a hipercaloric and fat rich diet. Interestingly, the Rubus supplement revert these changes although the mechanism of action remains to be elucidated.
The addition of Rubus extract to the CD produced some interesting results. The supplement decreased some fat depot accumulation in the rats fed the CD, although did not modify their low insulin sensitivity index. However, the intake of the Rubus extracts increased plasma TAG levels over the values found in rats of the SD group. This hypertriacyglycerolemic effect of the Rubus supplement can’t be related to any change in the LPL activity found in these animals when compared to those fed the SD, despite of the known effect of this enzyme controlling the clearance of circulating TAG [48,49]. The effect could be related to an increased production of TAG by the liver although the mechanism involved has not been established yet.
In view of the current results, it can be proposed that these effects of the Rubus extract supplement could contribute to reducing the impact on glucose metabolism in rats given the cafeteria diet as shown by the reduction in the AUCs for insulin without any changes in glucose levels observed in the OGTT.
As the main components of the Rubus extract are the flavonols and anthocyanins, it is reasonable to assume that the effects observed are due to these compounds, although small amounts of other phytochemicals are also present and may also be involved in the effects on health. In order to obtain reproducible results, extracts need to be obtained from the same plant material which has been standardized in terms of their contents of specific flavonols and anthocyanins, since the contents of these phytochemicals are known to fluctuate depending on environmental conditions [50]. Furthermore, absorption is subject to microfloral activity [9] so this also needs to be considered. Alternatively, studies using more purified extracts may yield more information about the active compounds responsible for these effects.
In conclusion, the study reported here not only demonstrates the obesogenic and metabolic effects of a cafeteria diet in female rats, but also shows that some of those effects are reduced when the same diet is supplemented with a Rubus extract. According to the current findings, it appears that such positive effects occur even without a significant change in insulin sensitivity, but additional research is needed to establish whether it also appears in rats fed the standard diet and to establish the mechanism involved.

Acknowledgements
The authors thank Milagros Morante for excellent technical help and pp-science-editing.com for editing and linguistic revision of the manuscript. Sources of financial support: Program of cooperation between Brazil and University San Pablo CEU sponsored by Airbus Military and Fundación Ramón Areces (CIVP16A1835) of Spain. AGL2009-08324, BES-2010-038057.

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