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Blackberry (Rubus sp. var. Lochness) extract reduces obesity induced by a cafeteria diet and affects the lipophilic metabolomic profile in rats
Kenia Bispo1, Marcel Piovezan2, Daniel Garc�a-Seco, Encarnaci�n Amusquivar, Danuta Dudzik, Beatriz Ramos-Solano, Javier Guti�rrez-Ma�ero, Coral Barbas and Emilio Herrera3 
Faculty of Pharmacy, Universidad San Pablo CEU, Madrid, Spain




Running head: Blackberry extract reduces cafeteria diet effects in rats 






1Affiliated to: CAPES Foundation, Ministry of Education of Brazil, Bras�lia � DF 70040-020, Brazil.
2Current address: Department of Chemistry, Federal University of Santa Catarina, Florianopolis, SC, Brazil 
3Corresponding autor: E. Herrera, Universidad San Pablo CEU, Ctra. Boadilla del Monte km 5.300, 28668-Boadilla del Monte (Madrid), Spain. Phone: +34 913724730; Fax: +34 913510496; email:  HYPERLINK "mailto:eherrera@ceu.es" eherrera@ceu.es. 
Abstract
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; the area under the curve of glucose and insulin after oral glucose load and inguinal adipose tissue LPL activity were also increased. Any of these variables were lower in rats of the CRD group, which also showed increased plasma triacylglycerols. However, both the CD and CRD decreased the insulin sensitivity index (ISI). The metabolomic variables showed that most of the acyl-carnitines were up-regulated whereas most of the phosphatidylcholines and lysophosphatidylcholines were down-regulated when comparing rats of the CD group versus those of SD, while when comparing CRD versus CD, oleic acid and lysophosphatidylethanolamines as well as phosphatidylserine and lysophosphatidylserine were up-regulated. 
In conclusion, besides evidencing the obesogenic and metabolic effects of a cafeteria diet in female rats, results show that such effects are reduced when the same diet is supplemented with this Rubus extract, although it did not modify the decreased the ISI values. 
Key words: Blackberry fruit extract � Adipose tissue � Insulin sensitivity � Lipoprotein lipase � Metabolomic profile � Rats


Introduction
Anthocyanins have several health benefits such as preventing cholesterol-induced atherosclerosis  ADDIN REFMGR.CITE <Refman><Cite><Author>Kadar</Author><Year>1979</Year><RecNum>32387</RecNum><IDText>Influence of anthocyanoside tratment on the cholesterol-induced atherosclerosis in the rabbit</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32387</Ref_ID><Title_Primary>Influence of anthocyanoside tratment on the cholesterol-induced atherosclerosis in the rabbit</Title_Primary><Authors_Primary>Kadar,A.</Authors_Primary><Authors_Primary>Robert,L.</Authors_Primary><Authors_Primary>Miskulin,M.</Authors_Primary><Authors_Primary>Tixier,J.M.</Authors_Primary><Authors_Primary>Brechemier,D.</Authors_Primary><Authors_Primary>Robert,A.M.</Authors_Primary><Date_Primary>1979</Date_Primary><Keywords>atherosclerosis</Keywords><Keywords>Rabbit</Keywords><Reprint>Not in File</Reprint><Start_Page>187</Start_Page><End_Page>205</End_Page><Periodical>Paroi Arterielle</Periodical><Volume>5</Volume><ZZ_JournalFull><f name="System">Paroi Arterielle</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(1), inhibiting platelet aggregation  ADDIN REFMGR.CITE <Refman><Cite><Author>Morazzoni</Author><Year>1996</Year><RecNum>32388</RecNum><IDText>Vaccinium myrtillus L</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32388</Ref_ID><Title_Primary>Vaccinium myrtillus L</Title_Primary><Authors_Primary>Morazzoni,P</Authors_Primary><Authors_Primary>Bombardelli,E.</Authors_Primary><Date_Primary>1996</Date_Primary><Keywords>Vaccinium myrtillus</Keywords><Keywords>l</Keywords><Reprint>Not in File</Reprint><Start_Page>3</Start_Page><End_Page>29</End_Page><Periodical>Fitoterapia</Periodical><Volume>67</Volume><ZZ_JournalFull><f name="System">Fitoterapia</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(2) and having antiinflammatory  ADDIN REFMGR.CITE <Refman><Cite><Author>Wang</Author><Year>1999</Year><RecNum>32389</RecNum><IDText>Antioxidant and anti-inflammatory activities of anthcyanins and their aglycon, cyanidin, from tart cherries</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32389</Ref_ID><Title_Primary>Antioxidant and anti-inflammatory activities of anthcyanins and their aglycon, cyanidin, from tart cherries</Title_Primary><Authors_Primary>Wang,H.</Authors_Primary><Authors_Primary>Nair,M.G.</Authors_Primary><Authors_Primary>Strasburg,G.M.</Authors_Primary><Authors_Primary>Chang,Y.-C.</Authors_Primary><Authors_Primary>Booren,A.M.</Authors_Primary><Authors_Primary>Gray,J.I.</Authors_Primary><Authors_Primary>DeWitt,D.L.</Authors_Primary><Date_Primary>1999</Date_Primary><Keywords>Antioxidant</Keywords><Keywords>antiinflammatory</Keywords><Keywords>Activity</Keywords><Reprint>Not in File</Reprint><Start_Page>296</Start_Page><Periodical>J.Nat.Prod.</Periodical><Volume>62</Volume><Issue>294</Issue><ZZ_JournalStdAbbrev><f name="System">J.Nat.Prod.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(3) and anticarcinogenic  ADDIN REFMGR.CITE <Refman><Cite><Author>Kamei</Author><Year>1995</Year><RecNum>32390</RecNum><IDText>Suppression of tumor cell growth by anthocyanins in vitro</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32390</Ref_ID><Title_Primary>Suppression of tumor cell growth by anthocyanins in vitro</Title_Primary><Authors_Primary>Kamei,H.</Authors_Primary><Authors_Primary>Kojima,T.</Authors_Primary><Authors_Primary>Hasegawa,M.</Authors_Primary><Authors_Primary>Koide,T.</Authors_Primary><Authors_Primary>Umeda,T.</Authors_Primary><Authors_Primary>Yokawa,T.</Authors_Primary><Authors_Primary>Terabe,K.</Authors_Primary><Date_Primary>1995</Date_Primary><Keywords>suppression</Keywords><Keywords>tumor</Keywords><Keywords>cell</Keywords><Keywords>cell growth</Keywords><Keywords>growth</Keywords><Keywords>anthocyanins</Keywords><Keywords>anthocyanin</Keywords><Keywords>in vitro</Keywords><Reprint>Not in File</Reprint><Start_Page>590</Start_Page><End_Page>594</End_Page><Periodical>Cancer Invest.</Periodical><Volume>13</Volume><ZZ_JournalStdAbbrev><f name="System">Cancer Invest.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(4) activities. Blackberry has a high content of phenolic compounds, which have been shown to inhibit oxidation of human LDL and lecithin liposomes  ADDIN REFMGR.CITE <Refman><Cite><Author>Heinonen</Author><Year>1998</Year><RecNum>506</RecNum><IDText>Antioxidant activity of berry phenolics on human low-density lipoprotein and liposome oxidation</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>506</Ref_ID><Title_Primary>Antioxidant activity of berry phenolics on human low-density lipoprotein and liposome oxidation</Title_Primary><Authors_Primary>Heinonen,I.M.</Authors_Primary><Authors_Primary>Meyer,A.S.</Authors_Primary><Authors_Primary>Frankel,E.N.</Authors_Primary><Date_Primary>1998</Date_Primary><Keywords>berries</Keywords><Keywords>antioxidants</Keywords><Keywords>LDL oxidation</Keywords><Keywords>liposomes</Keywords><Keywords>flavonoids</Keywords><Keywords>hydroxycinnamates</Keywords><Keywords>anthocyanins</Keywords><Keywords>flavan-3-ols</Keywords><Keywords>flavonols</Keywords><Keywords>antioxidant</Keywords><Keywords>antioxidant activity</Keywords><Keywords>human</Keywords><Keywords>low-density lipoprotein</Keywords><Keywords>lipoprotein</Keywords><Keywords>oxidation</Keywords><Keywords>lipoproteins</Keywords><Keywords>LDL</Keywords><Keywords>HPLC</Keywords><Reprint>Not in File</Reprint><Start_Page>4107</Start_Page><End_Page>4112</End_Page><Periodical>J.Agric.Food Chem.</Periodical><Volume>46</Volume><ZZ_JournalFull><f name="System">Journal of Agricultural and Food Chemistry</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Agric.Food Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(5) in vitro. Furthermore, blackberry anthocyanins suppress cancer cell growth by modifying cancer cell signaling pathways  ADDIN REFMGR.CITE <Refman><Cite><Author>Duthie</Author><Year>2007</Year><RecNum>32401</RecNum><IDText>Berry phytochemicals, genomic stability and cancer: evidence for chemoprotection at several stages in the carcinogenic process</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32401</Ref_ID><Title_Primary>Berry phytochemicals, genomic stability and cancer: evidence for chemoprotection at several stages in the carcinogenic process</Title_Primary><Authors_Primary>Duthie,S.J.</Authors_Primary><Date_Primary>2007</Date_Primary><Keywords>berry</Keywords><Keywords>phytochemicals</Keywords><Keywords>stability</Keywords><Keywords>cancer</Keywords><Keywords>chemoprotection</Keywords><Reprint>In File</Reprint><Start_Page>665</Start_Page><End_Page>674</End_Page><Periodical>Mol.Nutr.Food Res.</Periodical><Volume>51</Volume><ZZ_JournalStdAbbrev><f name="System">Mol.Nutr.Food Res.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(6, ADDIN REFMGR.CITE <Refman><Cite><Author>Tsuda</Author><Year>2003</Year><RecNum>3761</RecNum><IDText>Dietary cyanidin 3-O-b-D-glucoside-rich purple corn color prevents obesity and amellorates hyperglycemia in mice</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3761</Ref_ID><Title_Primary>Dietary cyanidin 3-O-b-D-glucoside-rich purple corn color prevents obesity and amellorates hyperglycemia in mice</Title_Primary><Authors_Primary>Tsuda,T.</Authors_Primary><Authors_Primary>Horio,F.</Authors_Primary><Authors_Primary>Uchida,K.</Authors_Primary><Authors_Primary>Apki,H.</Authors_Primary><Authors_Primary>Osawa,T.</Authors_Primary><Date_Primary>2003</Date_Primary><Keywords>DIETARY</Keywords><Keywords>COLOR</Keywords><Keywords>PREVENTS</Keywords><Keywords>obesity</Keywords><Keywords>hyperglycemia</Keywords><Reprint>In File</Reprint><Start_Page>2125</Start_Page><End_Page>2132</End_Page><Periodical>J.Nutr.</Periodical><Volume>133</Volume><ZZ_JournalFull><f name="System">Journal of Nutrition</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Nutr.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>7) and have been shown to improve body weight and body composition and to reduce obesity in mice  ADDIN REFMGR.CITE <Refman><Cite><Author>Prior</Author><Year>2008</Year><RecNum>3718</RecNum><IDText>Whole berries versus berry anthocyanins: interactions with dietary fat levels in the C57BL/6J mouse model of obesity</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3718</Ref_ID><Title_Primary>Whole berries versus berry anthocyanins: interactions with dietary fat levels in the C57BL/6J mouse model of obesity</Title_Primary><Authors_Primary>Prior,R.L.</Authors_Primary><Authors_Primary>Wu,X.</Authors_Primary><Authors_Primary>Gu,L.</Authors_Primary><Authors_Primary>Hager,T.J.</Authors_Primary><Authors_Primary>Hager,A.</Authors_Primary><Authors_Primary>Howard,L.R.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>DIETARY</Keywords><Keywords>dietary fat</Keywords><Keywords>DIETARY-FAT</Keywords><Keywords>fat</Keywords><Keywords>LEVEL</Keywords><Keywords>mouse</Keywords><Keywords>mouse model</Keywords><Keywords>model</Keywords><Keywords>obesity</Keywords><Reprint>Not in File</Reprint><Start_Page>647</Start_Page><End_Page>653</End_Page><Periodical>J.Agric.Food Chem.</Periodical><Volume>56</Volume><ZZ_JournalFull><f name="System">Journal of Agricultural and Food Chemistry</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Agric.Food Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(8). Nevertheless, the health benefits of blackberry have not been sufficiently explored  ADDIN REFMGR.CITE <Refman><Cite><Author>Kaume</Author><Year>2011</Year><RecNum>32400</RecNum><IDText>The blackberry fruit: a review on its composition and hemistry, metabolism and bioavailability, and health benefits</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32400</Ref_ID><Title_Primary>The blackberry fruit: a review on its composition and hemistry, metabolism and bioavailability, and health benefits</Title_Primary><Authors_Primary>Kaume,L.</Authors_Primary><Authors_Primary>Howard,L.R.</Authors_Primary><Authors_Primary>Devareddy,L.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>fruit</Keywords><Keywords>review</Keywords><Keywords>Composition</Keywords><Keywords>metabolism</Keywords><Keywords>bioavailability</Keywords><Keywords>health</Keywords><Keywords>benefits</Keywords><Reprint>In File</Reprint><Start_Page>5716</Start_Page><End_Page>5727</End_Page><Periodical>J.Agric.Food Chem.</Periodical><Volume>50</Volume><Web_URL_Link4><f name="System">Journal of Agricultural and Food Chemistry</f></Web_URL_Link4><ZZ_JournalStdAbbrev><f name="System">J.Agric.Food Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(9). 
Rodents fed a high-fat diet rapidly develop insulin resistance and impaired activation of the insulin-signaling pathway  ADDIN REFMGR.CITE <Refman><Cite><Author>Kim</Author><Year>2000</Year><RecNum>8610</RecNum><IDText>Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>8610</Ref_ID><Title_Primary>Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats</Title_Primary><Authors_Primary>Kim,C.H.</Authors_Primary><Authors_Primary>Youn,J.H.</Authors_Primary><Authors_Primary>Park,J.Y.</Authors_Primary><Authors_Primary>Hong,S.K.</Authors_Primary><Authors_Primary>Park,K.S.</Authors_Primary><Authors_Primary>Park,S.W.</Authors_Primary><Authors_Primary>Suh,K.I.</Authors_Primary><Authors_Primary>Lee,K.U.</Authors_Primary><Date_Primary>2000/6</Date_Primary><Keywords>glucose metabolic fluxes</Keywords><Keywords>glucose 6-phosphate</Keywords><Keywords>glucose transporter</Keywords><Keywords>hexokinase</Keywords><Keywords>glycogen synthase</Keywords><Keywords>effects</Keywords><Keywords>high-fat</Keywords><Keywords>high-fat diet</Keywords><Keywords>diet</Keywords><Keywords>exercise</Keywords><Keywords>exercise training</Keywords><Keywords>training</Keywords><Keywords>intracellular</Keywords><Keywords>glucose</Keywords><Keywords>glucose metabolism</Keywords><Keywords>metabolism</Keywords><Keywords>rats</Keywords><Keywords>rat</Keywords><Keywords>skeletal muscle</Keywords><Keywords>muscle</Keywords><Keywords>treatment</Keywords><Keywords>animals</Keywords><Keywords>hyperinsulinemic euglycemic clamp</Keywords><Keywords>clamp</Keywords><Keywords>GLUT-4</Keywords><Keywords>GLUT4</Keywords><Keywords>GLUT-4 protein</Keywords><Keywords>protein</Keywords><Keywords>glycogen</Keywords><Keywords>synthase</Keywords><Keywords>activities</Keywords><Keywords>glucose uptake</Keywords><Keywords>uptake</Keywords><Keywords>levels</Keywords><Keywords>cytosolic</Keywords><Keywords>insulin</Keywords><Keywords>insulin sensitivity</Keywords><Keywords>high-fat feeding</Keywords><Keywords>feeding</Keywords><Keywords>insulin resistance</Keywords><Keywords>insulin-resistance</Keywords><Keywords>resistance</Keywords><Keywords>glycolysis</Keywords><Keywords>glycogen synthesis</Keywords><Keywords>synthesis</Keywords><Keywords>glucose transport</Keywords><Keywords>transport</Keywords><Keywords>phosphorylation</Keywords><Reprint>Not in File</Reprint><Start_Page>E977</Start_Page><End_Page>E984</End_Page><Periodical>Am.J.Physiol.Endocrinol.Metab.</Periodical><Volume>278</Volume><Issue>6</Issue><ZZ_JournalFull><f name="System">American Journal of Physiology: Endocrinology and Metabolism</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Am.J.Physiol.Endocrinol.Metab.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Kraegen</Author><Year>1991</Year><RecNum>5696</RecNum><IDText>Development of muscle insulin resistance after liver insulin resistance in high-fat-fed rats</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>5696</Ref_ID><Title_Primary>Development of muscle insulin resistance after liver insulin resistance in high-fat-fed rats</Title_Primary><Authors_Primary>Kraegen,E.W.</Authors_Primary><Authors_Primary>Clark,P.W.</Authors_Primary><Authors_Primary>Jenkins,A.B.</Authors_Primary><Authors_Primary>Daley,E.A.</Authors_Primary><Authors_Primary>Chisholm,D.J.</Authors_Primary><Authors_Primary>Storlien,L.H.</Authors_Primary><Date_Primary>1991</Date_Primary><Keywords>development</Keywords><Keywords>muscle</Keywords><Keywords>muscle insulin resistance</Keywords><Keywords>insulin</Keywords><Keywords>insulin resistance</Keywords><Keywords>resistance</Keywords><Keywords>liver</Keywords><Keywords>rats</Keywords><Keywords>FFA</Keywords><Keywords>sensibilidad</Keywords><Keywords>insulina</Keywords><Keywords>m&#xFA;sculo</Keywords><Keywords>ramos</Keywords><Keywords>tesis</Keywords><Keywords>11.2.35</Keywords><Keywords>pilar</Keywords><Keywords>Tejido adiposo</Keywords><Reprint>In File</Reprint><Start_Page>1397</Start_Page><End_Page>1403</End_Page><Periodical>Diabetes</Periodical><Volume>40</Volume><ZZ_JournalFull><f name="System">Diabetes</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Todd</Author><Year>2007</Year><RecNum>2303</RecNum><IDText>Thiazolidinediones enhance skeletal muscle triacylglycerol synthesis while protecting against fatty acid-induced inflammation and insulin resistance</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>2303</Ref_ID><Title_Primary>Thiazolidinediones enhance skeletal muscle triacylglycerol synthesis while protecting against fatty acid-induced inflammation and insulin resistance</Title_Primary><Authors_Primary>Todd,M.K.</Authors_Primary><Authors_Primary>Watt,M.J.</Authors_Primary><Authors_Primary>Le,J.</Authors_Primary><Authors_Primary>Hevener,A.L.</Authors_Primary><Authors_Primary>Turcotte,L.P.</Authors_Primary><Date_Primary>2007</Date_Primary><Keywords>ACID</Keywords><Keywords>Article</Keywords><Keywords>CD36</Keywords><Keywords>ceramide</Keywords><Keywords>control</Keywords><Keywords>diacylglycerol</Keywords><Keywords>diet</Keywords><Keywords>DIETARY</Keywords><Keywords>DIFFERENTIAL REGULATION</Keywords><Keywords>fatty acid</Keywords><Keywords>feeding</Keywords><Keywords>glucose</Keywords><Keywords>HIGH-FAT</Keywords><Keywords>HYPOTHESIS</Keywords><Keywords>IKK-BETA</Keywords><Keywords>inflammation</Keywords><Keywords>inflammatory markers</Keywords><Keywords>insulin</Keywords><Keywords>insulin action</Keywords><Keywords>insulin action,lipid oversupply,nuclear factor-kappa B inflammatory pathway,lipotoxicity</Keywords><Keywords>insulin resistance</Keywords><Keywords>insulin sensitivity</Keywords><Keywords>INSULIN-RESISTANCE</Keywords><Keywords>JNK</Keywords><Keywords>KAPPA B ACTIVATION</Keywords><Keywords>kinetics</Keywords><Keywords>lipid</Keywords><Keywords>LIPID-LEVELS</Keywords><Keywords>MECHANISM</Keywords><Keywords>MEMBRANE</Keywords><Keywords>metabolism</Keywords><Keywords>muscle</Keywords><Keywords>phosphorylation</Keywords><Keywords>physiological</Keywords><Keywords>plasma</Keywords><Keywords>PLASMA-MEMBRANE</Keywords><Keywords>protein</Keywords><Keywords>PROTEIN-KINASE-C</Keywords><Keywords>rat</Keywords><Keywords>rats</Keywords><Keywords>RECEPTOR SUBSTRATE-1</Keywords><Keywords>RESISTANCE</Keywords><Keywords>rosiglitazone</Keywords><Keywords>SENSITIVITY</Keywords><Keywords>signaling</Keywords><Keywords>SIGNALING CASCADE</Keywords><Keywords>skeletal muscle</Keywords><Keywords>SKELETAL-MUSCLE</Keywords><Keywords>thiazolidinedione</Keywords><Keywords>thiazolidinediones</Keywords><Keywords>triacylglycerol</Keywords><Keywords>TRIACYLGLYCEROL SYNTHESIS</Keywords><Keywords>TRIGLYCERIDE CONTENT</Keywords><Keywords>troglitazone</Keywords><Keywords>uptake</Keywords><Keywords>WISTAR RATS</Keywords><Reprint>Not in File</Reprint><Start_Page>E485</Start_Page><End_Page>E493</End_Page><Periodical>American Journal of Physiology-Endocrinology and Metabolism</Periodical><Volume>292</Volume><Issue>2</Issue><ISSN_ISBN>0193-1849</ISSN_ISBN><Availability>Univ Calif San Diego, Dept Med, Div Endocrinol &amp; Metab, La Jolla, CA 92093 USA; Univ So Calif, Dept Kinesiol, Los Angeles, CA USA; Univ So Calif, Dept Biol Sci, Los Angeles, CA 90089 USA; Univ Melbourne, St Vincents Inst Med Res, Fitzroy, Vic 3065, Australia; Univ Melbourne, Dept Med, Fitzroy, Vic 3065, Australia</Availability><Web_URL>CCC:000243997100014</Web_URL><ZZ_JournalFull><f name="System">American Journal of Physiology-Endocrinology and Metabolism</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Akiyama</Author><Year>1996</Year><RecNum>19533</RecNum><IDText>High-fat hypercaloric diet induces obesity, glucose intolerance and hyperlipidemia in normal adult male Wistar rat</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>19533</Ref_ID><Title_Primary>High-fat hypercaloric diet induces obesity, glucose intolerance and hyperlipidemia in normal adult male Wistar rat</Title_Primary><Authors_Primary>Akiyama,T.</Authors_Primary><Authors_Primary>Tachibana,I.</Authors_Primary><Authors_Primary>Shirohara,H.</Authors_Primary><Authors_Primary>Watanabe,N.</Authors_Primary><Authors_Primary>Otsuki,M.</Authors_Primary><Date_Primary>1996</Date_Primary><Keywords>obesity</Keywords><Keywords>insulin resistance</Keywords><Keywords>High fat diet</Keywords><Keywords>hypercaloric diet</Keywords><Keywords>high fat</Keywords><Keywords>diet</Keywords><Keywords>glucose</Keywords><Keywords>glucose intolerance</Keywords><Keywords>Hyperlipidemia</Keywords><Keywords>adult</Keywords><Keywords>male</Keywords><Keywords>Wistar rat</Keywords><Keywords>rat</Keywords><Keywords>genetic</Keywords><Keywords>factors</Keywords><Keywords>development</Keywords><Keywords>humans</Keywords><Keywords>Animal</Keywords><Keywords>Energy</Keywords><Keywords>energy intake</Keywords><Keywords>intake</Keywords><Keywords>rats</Keywords><Keywords>obese</Keywords><Keywords>food</Keywords><Keywords>NO</Keywords><Keywords>Wistar rats</Keywords><Keywords>diabetic</Keywords><Keywords>high-fat diet</Keywords><Keywords>fat</Keywords><Keywords>body weight</Keywords><Keywords>weight</Keywords><Keywords>Glucose metabolism</Keywords><Keywords>metabolism</Keywords><Keywords>control</Keywords><Keywords>Stomach</Keywords><Keywords>gastric</Keywords><Keywords>fasting</Keywords><Keywords>serum</Keywords><Keywords>concentration</Keywords><Keywords>aspartate aminotransferase</Keywords><Keywords>alanine</Keywords><Keywords>alanine aminotransferase</Keywords><Keywords>total cholesterol</Keywords><Keywords>cholesterol</Keywords><Keywords>Triglyceride</Keywords><Keywords>Phospholipid</Keywords><Keywords>free fatty</Keywords><Keywords>free fatty acids</Keywords><Keywords>fatty acids</Keywords><Keywords>acids</Keywords><Keywords>FFA</Keywords><Keywords>glucose loading</Keywords><Keywords>weight gain</Keywords><Keywords>compared</Keywords><Keywords>liver</Keywords><Keywords>liver weight</Keywords><Keywords>steatosis</Keywords><Keywords>pancreas</Keywords><Keywords>protein</Keywords><Keywords>amylase</Keywords><Keywords>ALL</Keywords><Keywords>DNA</Keywords><Keywords>Serum glucose</Keywords><Keywords>insulin</Keywords><Keywords>insulin response</Keywords><Keywords>response</Keywords><Keywords>relatives</Keywords><Keywords>glucose response</Keywords><Keywords>resistance</Keywords><Keywords>feeding</Keywords><Keywords>hyperinsulinemia</Keywords><Reprint>Not in File</Reprint><Start_Page>27</Start_Page><End_Page>35</End_Page><Periodical>Diabetes Res.Clin.Pract.</Periodical><Volume>31</Volume><ZZ_JournalFull><f name="System">Diabetes Research and Clinical Practice</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Diabetes Res.Clin.Pract.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(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  ADDIN REFMGR.CITE <Refman><Cite><Author>Kiens</Author><Year>1996</Year><RecNum>16764</RecNum><IDText>Types of carbohydrate in an ordinary diet affect insulin action and muscle substrates in humans</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>16764</Ref_ID><Title_Primary>Types of carbohydrate in an ordinary diet affect insulin action and muscle substrates in humans</Title_Primary><Authors_Primary>Kiens,B.</Authors_Primary><Authors_Primary>Richter,E.A.</Authors_Primary><Date_Primary>1996</Date_Primary><Keywords>insulin sensitivity</Keywords><Keywords>Dietary fiber</Keywords><Keywords>muscle</Keywords><Keywords>glycogen</Keywords><Keywords>Triacylglycerols</Keywords><Keywords>glycemic index</Keywords><Keywords>carbohydrate</Keywords><Keywords>diet</Keywords><Keywords>insulin</Keywords><Keywords>insulin action</Keywords><Keywords>humans</Keywords><Keywords>dietary</Keywords><Keywords>dietary carbohydrate</Keywords><Keywords>men</Keywords><Keywords>diets</Keywords><Keywords>Energy</Keywords><Keywords>Carbohydrates</Keywords><Keywords>fat</Keywords><Keywords>protein</Keywords><Keywords>index</Keywords><Keywords>euglycemic</Keywords><Keywords>euglycemic hyperinsulinemic clamp</Keywords><Keywords>hyperinsulinemic clamp</Keywords><Keywords>clamp</Keywords><Keywords>glucose</Keywords><Keywords>glucose uptake</Keywords><Keywords>uptake</Keywords><Keywords>Plasma</Keywords><Keywords>Plasma insulin</Keywords><Keywords>concentration</Keywords><Keywords>compared</Keywords><Keywords>Plasma fatty acids</Keywords><Keywords>fatty acid</Keywords><Keywords>acid</Keywords><Keywords>blood</Keywords><Keywords>Blood glucose</Keywords><Keywords>difference</Keywords><Keywords>muscle glycogen</Keywords><Keywords>triacylglycerol</Keywords><Keywords>Composition</Keywords><Keywords>whole-body glucose disposal</Keywords><Keywords>glucose disposal</Keywords><Keywords>disposal</Keywords><Keywords>adaptation</Keywords><Keywords>term</Keywords><Keywords>digestion</Keywords><Keywords>absorption</Keywords><Reprint>Not in File</Reprint><Start_Page>47</Start_Page><End_Page>53</End_Page><Periodical>Am.J.Clin.Nutr.</Periodical><Volume>63</Volume><ZZ_JournalFull><f name="System">American Journal of Clinical Nutrition</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Am.J.Clin.Nutr.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(14). The cafeteria diet � composed of high-fat and high-sugar supermarket products � results in obesity, glucose intolerance and insulin resistance in rats  ADDIN REFMGR.CITE <Refman><Cite><Author>Petry</Author><Year>1997</Year><RecNum>583</RecNum><IDText>Early and late nutritional windows for diabetes susceptibility</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>583</Ref_ID><Title_Primary>Early and late nutritional windows for diabetes susceptibility</Title_Primary><Authors_Primary>Petry,C.J.</Authors_Primary><Authors_Primary>Desal,M.</Authors_Primary><Authors_Primary>Ozanne,S.E.</Authors_Primary><Authors_Primary>Hales,C.N.</Authors_Primary><Date_Primary>1997</Date_Primary><Keywords>diabetes</Keywords><Reprint>In File</Reprint><Start_Page>233</Start_Page><End_Page>242</End_Page><Periodical>Proc.Nutr.Soc.</Periodical><Volume>56</Volume><ZZ_JournalStdAbbrev><f name="System">Proc.Nutr.Soc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Brandt</Author><Year>2010</Year><RecNum>584</RecNum><IDText>Cafeteria diet-induced insulin resistance is not associated with decreased insulin signaling or AMPK activity and is alleviated by physical training in rats</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>584</Ref_ID><Title_Primary>Cafeteria diet-induced insulin resistance is not associated with decreased insulin signaling or AMPK activity and is alleviated by physical training in rats</Title_Primary><Authors_Primary>Brandt,N.</Authors_Primary><Authors_Primary>De Bock,K.</Authors_Primary><Authors_Primary>Richter,E.A.</Authors_Primary><Authors_Primary>Hespel,P.</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>insulin</Keywords><Keywords>insulin resistance</Keywords><Keywords>AMPK</Keywords><Keywords>rats</Keywords><Keywords>rat</Keywords><Reprint>In File</Reprint><Start_Page>E215</Start_Page><End_Page>E224</End_Page><Periodical>Am.J.Physiol.Endocrinol.Metab.</Periodical><Volume>299</Volume><ZZ_JournalFull><f name="System">American Journal of Physiology: Endocrinology and Metabolism</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Am.J.Physiol.Endocrinol.Metab.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(15,16) and hamsters  ADDIN REFMGR.CITE <Refman><Cite><Author>Carrillon</Author><Year>2013</Year><RecNum>586</RecNum><IDText>Cafeteria diet induces obesity and insulin resistance associated with oxidative stress but not with inflammation: improvement by dietary supplementation with a melon superoxide dismutase</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>586</Ref_ID><Title_Primary>Cafeteria diet induces obesity and insulin resistance associated with oxidative stress but not with inflammation: improvement by dietary supplementation with a melon superoxide dismutase</Title_Primary><Authors_Primary>Carrillon,J.</Authors_Primary><Authors_Primary>Romain,C.</Authors_Primary><Authors_Primary>Bardy,G.</Authors_Primary><Authors_Primary>Fouret,G.</Authors_Primary><Authors_Primary>Feillet-Coudray,C.</Authors_Primary><Authors_Primary>Gaillet,S.</Authors_Primary><Authors_Primary>Lacan,D.</Authors_Primary><Authors_Primary>Cristol,J.P.</Authors_Primary><Authors_Primary>Rouanet,J.M.</Authors_Primary><Date_Primary>2013</Date_Primary><Keywords>diet</Keywords><Keywords>obesity</Keywords><Keywords>insulin</Keywords><Keywords>insulin resistance</Keywords><Keywords>oxidative stress</Keywords><Keywords>inflammation</Keywords><Keywords>superoxide</Keywords><Reprint>In File</Reprint><Start_Page>254</Start_Page><End_Page>261</End_Page><Periodical>Free Radic.Biol.Med.</Periodical><Volume>65</Volume><ZZ_JournalFull><f name="System">Free Radical Biology and Medicine</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Free Radic.Biol.Med.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(17), and reduced insulin clearance in mice  ADDIN REFMGR.CITE <Refman><Cite><Author>Brandimarti</Author><Year>2013</Year><RecNum>585</RecNum><IDText>Cafeteria diet inhibits insulin clearance by reduced insulin-degrading enzyme expression and mRNA splicing</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>585</Ref_ID><Title_Primary>Cafeteria diet inhibits insulin clearance by reduced insulin-degrading enzyme expression and mRNA splicing</Title_Primary><Authors_Primary>Brandimarti,P.</Authors_Primary><Authors_Primary>Costa-J&#xFA;nior,J.M.</Authors_Primary><Authors_Primary>Ferreira,S.M.</Authors_Primary><Authors_Primary>Protzek,A.O.</Authors_Primary><Authors_Primary>Santos,G.J.</Authors_Primary><Authors_Primary>Carneiro,E.M.</Authors_Primary><Authors_Primary>Boschero,A.C.</Authors_Primary><Authors_Primary>Rezende,L.F.</Authors_Primary><Date_Primary>2013</Date_Primary><Keywords>diet</Keywords><Keywords>insulin</Keywords><Keywords>insulin clearance</Keywords><Keywords>enzyme</Keywords><Reprint>In File</Reprint><Start_Page>173</Start_Page><End_Page>182</End_Page><Periodical>J.Endocrinol.</Periodical><Volume>219</Volume><ZZ_JournalFull><f name="System">Journal of Endocrinology</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Endocrinol.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(18), and has been considered a robust model of metabolic syndrome in humans  ADDIN REFMGR.CITE <Refman><Cite><Author>Sampey</Author><Year>2011</Year><RecNum>3658</RecNum><IDText>Cafeteria diet is a robust model of human metabolic syndrome with liver and adipose tissue inflammation: comparison to high-fat diet</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3658</Ref_ID><Title_Primary>Cafeteria diet is a robust model of human metabolic syndrome with liver and adipose tissue inflammation: comparison to high-fat diet</Title_Primary><Authors_Primary>Sampey,B.P.</Authors_Primary><Authors_Primary>Vanhoose,A.M.</Authors_Primary><Authors_Primary>Winfield,H.M.</Authors_Primary><Authors_Primary>Freemerman,A.J.</Authors_Primary><Authors_Primary>Muehlbauer,M.J.</Authors_Primary><Authors_Primary>Fueger,P.T.</Authors_Primary><Authors_Primary>Newgard,C.B.</Authors_Primary><Authors_Primary>Makowski,L.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>diet</Keywords><Keywords>model</Keywords><Keywords>human</Keywords><Keywords>metabolic</Keywords><Keywords>metabolic syndrome</Keywords><Keywords>syndrome</Keywords><Keywords>liver</Keywords><Keywords>ADIPOSE</Keywords><Keywords>adipose tissue</Keywords><Keywords>ADIPOSE-TISSUE</Keywords><Keywords>TISSUE</Keywords><Keywords>inflammation</Keywords><Keywords>HIGH-FAT</Keywords><Keywords>high-fat diet</Keywords><Reprint>Not in File</Reprint><Periodical>Obesity</Periodical><Volume>doi:10.1038/oby.2011.18</Volume><ZZ_JournalFull><f name="System">Obesity</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(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  ADDIN REFMGR.CITE <Refman><Cite><Author>Legendre</Author><Year>1998</Year><RecNum>593</RecNum><IDText>Numerical Ecology</IDText><MDL Ref_Type="Book, Whole"><Ref_Type>Book, Whole</Ref_Type><Ref_ID>593</Ref_ID><Title_Primary>Numerical Ecology</Title_Primary><Authors_Primary>Legendre,P.</Authors_Primary><Authors_Primary>Legendre,L.</Authors_Primary><Date_Primary>1998</Date_Primary><Reprint>In File</Reprint><Volume>2nd</Volume><Authors_Secondary>Elsevier,Science B.V.</Authors_Secondary><Pub_Place>Amsterdam</Pub_Place><ZZ_WorkformID>2</ZZ_WorkformID></MDL></Cite></Refman>(20). The benefits of a holistic approach using metabolomics may result in identification of new metabolic markers to predict the development of some diseases  ADDIN REFMGR.CITE <Refman><Cite><Author>Ciborowski</Author><Year>2012</Year><RecNum>594</RecNum><IDText>Metabolomics with LC-QTOF-MS permits the prediction of disease stage in aortic abdominal aneurysm based on plasma metabolic fingerprint</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>594</Ref_ID><Title_Primary>Metabolomics with LC-QTOF-MS permits the prediction of disease stage in aortic abdominal aneurysm based on plasma metabolic fingerprint</Title_Primary><Authors_Primary>Ciborowski,M.</Authors_Primary><Authors_Primary>Teul,J.</Authors_Primary><Authors_Primary>Martin-Ventura,J.L.</Authors_Primary><Authors_Primary>Egido,J.</Authors_Primary><Authors_Primary>Barbas,C.</Authors_Primary><Date_Primary>2012</Date_Primary><Keywords>metabolomics</Keywords><Keywords>plasma</Keywords><Reprint>In File</Reprint><Periodical>PloS One</Periodical><Volume>DOI: 10.1371/journal.pone.0031982</Volume><ZZ_JournalFull><f name="System">PloS One</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(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  ADDIN REFMGR.CITE <Refman><Cite><Author>Estrany</Author><Year>2012</Year><RecNum>15022</RecNum><IDText>Isocaloric intake of a high-fat diet modifies adiposity and lipid handling in a sex dependent manner in rats</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>15022</Ref_ID><Title_Primary>Isocaloric intake of a high-fat diet modifies adiposity and lipid handling in a sex dependent manner in rats</Title_Primary><Authors_Primary>Estrany,M.E.</Authors_Primary><Authors_Primary>Proenza,A.M.</Authors_Primary><Authors_Primary>Llad&#xF3;,I</Authors_Primary><Authors_Primary>Gianotti,M.</Authors_Primary><Date_Primary>2012</Date_Primary><Keywords>intake</Keywords><Keywords>high-fat</Keywords><Keywords>high-fat diet</Keywords><Keywords>high fat diet</Keywords><Keywords>diet</Keywords><Keywords>adiposity</Keywords><Keywords>lipid</Keywords><Keywords>sex</Keywords><Keywords>rats</Keywords><Keywords>rat</Keywords><Reprint>Not in File</Reprint><Start_Page>52</Start_Page><End_Page>62</End_Page><Periodical>Lipids in Health and Disease</Periodical><Volume>10</Volume><ZZ_JournalFull><f name="System">Lipids in Health and Disease</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(22) and cafeteria diet  ADDIN REFMGR.CITE <Refman><Cite><Author>Llad&#xF3;</Author><Year>2000</Year><RecNum>11996</RecNum><IDText>Effects of cafeteria diet feeding on b3-adrenoceptor expression and lipolytic activity in white adipose tissue of male and female rats</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>11996</Ref_ID><Title_Primary>Effects of cafeteria diet feeding on <f name="Symbol">b</f><sub>3</sub>-adrenoceptor expression and lipolytic activity in white adipose tissue of male and female rats</Title_Primary><Authors_Primary>Llad&#xF3;,I</Authors_Primary><Authors_Primary>Estrany,M.E.</Authors_Primary><Authors_Primary>Rodr&#xED;guez,E.</Authors_Primary><Authors_Primary>Amengual,B.</Authors_Primary><Authors_Primary>Roca,P.</Authors_Primary><Authors_Primary>Palou,A.</Authors_Primary><Date_Primary>2000/11</Date_Primary><Keywords>cafeteria diet feeding</Keywords><Keywords><f name="Symbol">b</f>(3)-AR</Keywords><Keywords>lipolytic activity</Keywords><Keywords>CGP12177A</Keywords><Keywords>sex</Keywords><Keywords>rat</Keywords><Keywords>effects</Keywords><Keywords>cafeteria diet</Keywords><Keywords>diet</Keywords><Keywords>feeding</Keywords><Keywords>expression</Keywords><Keywords>activity</Keywords><Keywords>activities</Keywords><Keywords>white adipose tissue</Keywords><Keywords>adipose tissue</Keywords><Keywords>female</Keywords><Keywords>female rats</Keywords><Keywords>rats</Keywords><Keywords>in vivo</Keywords><Keywords>stimulation</Keywords><Keywords>white adipocytes</Keywords><Keywords>adipocytes</Keywords><Keywords>adipocyte</Keywords><Keywords>animals</Keywords><Keywords>Wistar rat</Keywords><Keywords>age</Keywords><Keywords>measurement</Keywords><Keywords>leptin</Keywords><Keywords>in vitro</Keywords><Keywords>in vitro study</Keywords><Keywords>Study</Keywords><Keywords>control</Keywords><Keywords>male rats</Keywords><Keywords>male rat</Keywords><Keywords>levels</Keywords><Keywords>leptin mRNA</Keywords><Keywords>mRNA</Keywords><Keywords>noradrenaline</Keywords><Keywords>lipolysis</Keywords><Keywords>fat</Keywords><Keywords>fat cells</Keywords><Keywords>fat cell</Keywords><Keywords>cells</Keywords><Keywords>females</Keywords><Keywords>glycerol</Keywords><Keywords>NO</Keywords><Keywords>treatment</Keywords><Keywords>forskolin</Keywords><Keywords>cyclic AMP</Keywords><Keywords>cyclic-AMP</Keywords><Keywords>body weight</Keywords><Keywords>ARE</Keywords><Keywords>Se</Keywords><Keywords>adrenoceptor</Keywords><Reprint>Not in File</Reprint><Start_Page>1396</Start_Page><End_Page>1404</End_Page><Periodical>Int.J.Obes.</Periodical><Volume>24</Volume><Issue>11</Issue><ZZ_JournalFull><f name="System">International Journal of Obesity</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Int.J.Obes.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(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  ADDIN REFMGR.CITE <Refman><Cite><Author>Garc&#xED;a-Seco</Author><Year>2012</Year><RecNum>15029</RecNum><IDText>Enhanced blackberry production using Pseudomonas fluorescents as elicitor</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>15029</Ref_ID><Title_Primary>Enhanced blackberry production using Pseudomonas fluorescents as elicitor</Title_Primary><Authors_Primary>Garc&#xED;a-Seco,D.</Authors_Primary><Authors_Primary>Bonilla,A.</Authors_Primary><Authors_Primary>Algar,E.</Authors_Primary><Authors_Primary>Garc&#xED;a-Villaraco,A.</Authors_Primary><Authors_Primary>Gutierrez-Ma&#xF1;ero,J.</Authors_Primary><Authors_Primary>Ramos-Solano,B.</Authors_Primary><Date_Primary>2012</Date_Primary><Reprint>In File</Reprint><Periodical>Agron.Sustain.Dev.</Periodical><Volume>DOI 10.1007/s13593-012-0103-z</Volume><ZZ_JournalStdAbbrev><f name="System">Agron.Sustain.Dev.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(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  ADDIN REFMGR.CITE <Refman><Cite><Author>Giusti</Author><Year>2011</Year><RecNum>32395</RecNum><IDText>Anthocyanins. Characterization and measurement with UV-visible spectroscopy</IDText><MDL Ref_Type="Book Chapter"><Ref_Type>Book Chapter</Ref_Type><Ref_ID>32395</Ref_ID><Title_Primary>Anthocyanins. Characterization and measurement with UV-visible spectroscopy</Title_Primary><Authors_Primary>Giusti,M.M.</Authors_Primary><Authors_Primary>Wrolstad,R.E.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>anthocyanins</Keywords><Keywords>anthocyanin</Keywords><Keywords>characterization</Keywords><Keywords>measurement</Keywords><Keywords>spectroscopy</Keywords><Keywords>food</Keywords><Reprint>In File</Reprint><Title_Secondary>Current protocols in Food Analytical Chemistry</Title_Secondary><Authors_Secondary>Wrostad,R.E.</Authors_Secondary><Authors_Secondary>Acree,T.E.</Authors_Secondary><Authors_Secondary>An,H.</Authors_Secondary><Authors_Secondary>Decker,E.A.</Authors_Secondary><Authors_Secondary>Penner,M.H.</Authors_Secondary><Authors_Secondary>Reid,D.S.</Authors_Secondary><Authors_Secondary>Schwatz,S.J.</Authors_Secondary><Authors_Secondary>Shoemaker,C.F.</Authors_Secondary><Authors_Secondary>Spoms,P.</Authors_Secondary><Pub_Place>New York</Pub_Place><Publisher>John Wiley and Sons</Publisher><ZZ_WorkformID>3</ZZ_WorkformID></MDL></Cite></Refman>(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  ADDIN REFMGR.CITE <Refman><Cite><Author>Zhishen</Author><Year>2013</Year><RecNum>32391</RecNum><IDText>The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32391</Ref_ID><Title_Primary>The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals</Title_Primary><Authors_Primary>Zhishen,J.</Authors_Primary><Authors_Primary>Mengcheng,T.</Authors_Primary><Authors_Primary>Jianming,W.</Authors_Primary><Date_Primary>2013</Date_Primary><Keywords>Flavonoid</Keywords><Keywords>scavenging</Keywords><Keywords>scavenging effect</Keywords><Keywords>superoxide</Keywords><Keywords>superoxide radicals</Keywords><Keywords>Superoxide radical</Keywords><Keywords>radicals</Keywords><Keywords>radical</Keywords><Reprint>Not in File</Reprint><Start_Page>555</Start_Page><End_Page>559</End_Page><Periodical>Food Chem.</Periodical><Volume>64</Volume><ZZ_JournalStdAbbrev><f name="System">Food Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(26) using catechin (Sigma Chemical Co., St. Louis, MO) as standard; (-)epicatechin was the predominant flavonoid as described in  ADDIN REFMGR.CITE <Refman><Cite><Author>Piovezan</Author><Year>2013</Year><RecNum>3749</RecNum><IDText>Method development for determination of (+)-catechin and (-)epicatechin by micellar electrokinetic chromatography (MEKC). Annual characterzation of field grown blackberries</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3749</Ref_ID><Title_Primary>Method development for determination of (+)-catechin and (-)epicatechin by micellar electrokinetic chromatography (MEKC). Annual characterzation of field grown blackberries</Title_Primary><Authors_Primary>Piovezan,M.</Authors_Primary><Authors_Primary>Garc&#xED;a-Seco,D.</Authors_Primary><Authors_Primary>Micke,G.A.</Authors_Primary><Authors_Primary>Guti&#xE9;rrez-Ma&#xF1;ero,J.</Authors_Primary><Authors_Primary>Ramos-Solano,B.</Authors_Primary><Date_Primary>2013</Date_Primary><Keywords>development</Keywords><Keywords>CHROMATOGRAPHY</Keywords><Reprint>Not in File</Reprint><Start_Page>2251</Start_Page><End_Page>2258</End_Page><Periodical>Electrophoresis</Periodical><Volume>34</Volume><ZZ_JournalFull><f name="System">Electrophoresis</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(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. Composition and fatty acid profile of the diets are shown in Tables 1 and 2 respectively. The CD and CRD were stored at -20�C 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 4�C and stored at -80�C until analyzed for glucose and insulin. The insulin sensitivity index (ISI) was calculated as previously described  ADDIN REFMGR.CITE <Refman><Cite><Author>Cacho</Author><Year>2008</Year><RecNum>3500</RecNum><IDText>Validation of simple indexes to asses insulin sensitivity during pregnancy in Wistar and Sprague Dawley rats</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3500</Ref_ID><Title_Primary>Validation of simple indexes to asses insulin sensitivity during pregnancy in Wistar and Sprague Dawley rats</Title_Primary><Authors_Primary>Cacho,J.</Authors_Primary><Authors_Primary>Sevillano,J.</Authors_Primary><Authors_Primary>de Castro,J.</Authors_Primary><Authors_Primary>Herrera,E.</Authors_Primary><Authors_Primary>Ramos,M.P.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>INDEXES</Keywords><Keywords>INDEX</Keywords><Keywords>insulin</Keywords><Keywords>insulin sensitivity</Keywords><Keywords>SENSITIVITY</Keywords><Keywords>pregnancy</Keywords><Keywords>PREGNANCIES</Keywords><Keywords>rats</Keywords><Keywords>rat</Keywords><Reprint>On Request 06/02/2012</Reprint><Start_Page>E1269</Start_Page><End_Page>E1276</End_Page><Periodical>Am.J.Physiol.Endocrinol.Metab.</Periodical><Volume>295</Volume><ZZ_JournalFull><f name="System">American Journal of Physiology: Endocrinology and Metabolism</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Am.J.Physiol.Endocrinol.Metab.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(28) 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 �L/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 -80�C 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  ADDIN REFMGR.CITE <Refman><Cite><Author>Folch</Author><Year>1957</Year><RecNum>7648</RecNum><IDText>A simple method for the isolation and purification of total lipids from animal tissues</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>7648</Ref_ID><Title_Primary>A simple method for the isolation and purification of total lipids from animal tissues</Title_Primary><Authors_Primary>Folch,J.</Authors_Primary><Authors_Primary>Lees,M.</Authors_Primary><Authors_Primary>Sloane Stanley,G.H.</Authors_Primary><Date_Primary>1957</Date_Primary><Keywords>method</Keywords><Keywords>Purification</Keywords><Keywords>lipids</Keywords><Keywords>Tissues</Keywords><Reprint>In File</Reprint><Start_Page>24</Start_Page><End_Page>36</End_Page><Periodical>J.Biol.Chem.</Periodical><Volume>22</Volume><ZZ_JournalFull><f name="System">Journal of Biological Chemistry</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Biol.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(29). 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 80�C for 2.5�h as previously described  ADDIN REFMGR.CITE <Refman><Cite><Author>Amusquivar</Author><Year>2011</Year><RecNum>3703</RecNum><IDText>Evaluation of two methods for plasma fatty acid analysis by GC</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3703</Ref_ID><Title_Primary>Evaluation of two methods for plasma fatty acid analysis by GC</Title_Primary><Authors_Primary>Amusquivar,E.</Authors_Primary><Authors_Primary>Schiffner,S.</Authors_Primary><Authors_Primary>Herrera,E.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>plasma</Keywords><Keywords>fatty acid</Keywords><Keywords>FATTY-ACID</Keywords><Keywords>ACID</Keywords><Keywords>analysis</Keywords><Reprint>Not in File</Reprint><Start_Page>711</Start_Page><End_Page>716</End_Page><Periodical>Eur.J.Lipid Sci.Technol.</Periodical><Volume>113</Volume><Web_URL_Link4><f name="System">Eur.J.Lipid Sci.Technol.</f></Web_URL_Link4><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(30). 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  ADDIN REFMGR.CITE <Refman><Cite><Author>Llobera</Author><Year>1979</Year><RecNum>1063</RecNum><IDText>Lipoprotein lipase activity in liver of the rat fetus</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>1063</Ref_ID><Title_Primary>Lipoprotein lipase activity in liver of the rat fetus</Title_Primary><Authors_Primary>Llobera,M.</Authors_Primary><Authors_Primary>Montes,A.</Authors_Primary><Authors_Primary>Herrera,E.</Authors_Primary><Date_Primary>1979</Date_Primary><Keywords>lipoprotein</Keywords><Keywords>lipase</Keywords><Keywords>liver</Keywords><Keywords>rat</Keywords><Keywords>fetus</Keywords><Reprint>Not in File</Reprint><Start_Page>272</Start_Page><End_Page>277</End_Page><Periodical>Biochem.Biophys.Res.Commun.</Periodical><Volume>91</Volume><ZZ_JournalFull><f name="System">Biochemical and Biophysical Research Communications</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">Biochem.Biophys.Res.Commun.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(31) 
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 �m nylon filter. Quality control (QC) was determined  ADDIN REFMGR.CITE <Refman><Cite><Author>Gika</Author><Year>2008</Year><RecNum>3760</RecNum><IDText>Evaluation of the repeatability of ultra-performance liquid chromatography-TOF-MS for global metabolic profiling of human urine samples</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3760</Ref_ID><Title_Primary>Evaluation of the repeatability of ultra-performance liquid chromatography-TOF-MS for global metabolic profiling of human urine samples</Title_Primary><Authors_Primary>Gika,H.</Authors_Primary><Authors_Primary>Macpherson,E.</Authors_Primary><Authors_Primary>Theodoridis,G.</Authors_Primary><Authors_Primary>Wilson,I.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>metabolic</Keywords><Keywords>human</Keywords><Keywords>SAMPLE</Keywords><Reprint>In File</Reprint><Start_Page>299</Start_Page><End_Page>305</End_Page><Periodical>J.Chromatogr.B Biomed.Sci.Appl.</Periodical><Volume>871</Volume><ZZ_JournalFull><f name="System">Journal of Chromatography B: Biomedical Sciences and Applications</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Chromatogr.B Biomed.Sci.Appl.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(32) 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��L of extracted plasma samples that were injected to a reverse-phase Zorbax Extend C18 column (2.1 � 50 mm, 1.8 �m, Agilent Technologies) at 60�C. 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 250�C 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 .
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). One-way analysis of variance (ANOVA) was used to compare different diets. When treatment effects were significantly different (p < 0.05), Newman-Keuls simultaneous tests were used to establish statistical differences between individual dietary interventions. 
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, filtering and statistical analysis were performed by Mass Profiler Professional (MPP, version B.12.1, Agilent Technologies) software. Statistical analysis after filtering by samples frequency according to specified comparisons was performed using unpaired t-test (p < 0.05), assuming unequal variance (Welch�s t-test). The multivariate analysis, statistical calculations and plottings were obtained with SIMCA P+ 12.0 (Umetrics, Umea, Sweden). 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.
Results 
Metabolic changes: 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 polyunsaturated fatty acids (PUFA), mainly corresponding to linoleic acid (18:2 n-6), is similar (Table 2). Total food intake by rats of all three groups was similar throughout all the experiment, meaning that (due to the higher energy content of the cafeteria diets) the energy intake of rats in groups CD and CRD was higher than those in the SD group (data not shown). 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 difference between the groups was found in the plasma concentrations of either stearic acid (18:0) or any of the PUFA (Table 3). 
Body, liver and different fat depot weights and plasma metabolic variables of the rats are shown in Table 4. The consumption of CD slightly, but not significantly, increased body weight, the effect disappearing in those rats fed the CRD; liver weight was higher in the CD group than in SD (p < 0.05), the effect again 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 (see Table 4). Plasma triacylglycerols were higher in those rats fed the CD and CRD than those on 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 of the CD group than in those of SD; once again this variable decreased in rats of 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. 
Metabolomic variables: The common statistical approach used in metabolomics data analysis is based on 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  ADDIN REFMGR.CITE <Refman><Cite><Author>Gika</Author><Year>2008</Year><RecNum>32402</RecNum><IDText>Evaluation of the repeatability of ultra-performance liquid chromatography-TOF-MS for global metabolic profiling of human urine samples</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32402</Ref_ID><Title_Primary>Evaluation of the repeatability of ultra-performance liquid chromatography-TOF-MS for global metabolic profiling of human urine samples</Title_Primary><Authors_Primary>Gika,H.G.</Authors_Primary><Authors_Primary>Macpherson,E.</Authors_Primary><Authors_Primary>Theodoridis,G.A.</Authors_Primary><Authors_Primary>Wilson,I.D.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>evaluation</Keywords><Keywords>repeatability</Keywords><Keywords>human</Keywords><Keywords>urine</Keywords><Reprint>In File</Reprint><Start_Page>299</Start_Page><End_Page>305</End_Page><Periodical>Anal.Technol.Biomed.Life Sc.</Periodical><Volume>871</Volume><ZZ_JournalStdAbbrev><f name="System">Anal.Technol.Biomed.Life Sc.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(33). The coefficient of variation (% CV) of QC samples was calculated and values are shown in Tables 6 and 7.  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 1 (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 (PCA-X) of the results was employed. In the supervised models, a better separation among the three groups (SD, CD and CDR) is seen (Figure 1, 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. Figures 2 and 3 show all the different Partial Least Squares Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) models for the comparisons and Tables 6 and 7 summarize the most relevant changes found in the univariate and multivariate analyses corresponding to statistically significant differences of identified compounds when comparing those from CD vs SD and CD vs CDR groups. When comparing rats of the CD group vs those of SD (Table 6), it was found that oleic acid, phosphocholine and most of the acylcarnitine derivatives were up-regulated in the CD group, whereas most phophadidylcholines and lysophosphadidylcholines (lysoPC) were down-regulated. However, when comparing rats of the CRD group versus those of the CD (Table 7) it appears that oleic acid and lysophosphatidylethanolamine (lysoPE) were up-regulated in the CDR group, whereas lysoPCs and ceramide-phosphate (cerP) were down-regulated.
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 so-called cafeteria diet) to young female rats corresponds to a diet-induced obesity that mimics metabolic syndrome in humans, in agreement with a previous proposal  ADDIN REFMGR.CITE <Refman><Cite><Author>Sampey</Author><Year>2011</Year><RecNum>3658</RecNum><IDText>Cafeteria diet is a robust model of human metabolic syndrome with liver and adipose tissue inflammation: comparison to high-fat diet</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>3658</Ref_ID><Title_Primary>Cafeteria diet is a robust model of human metabolic syndrome with liver and adipose tissue inflammation: comparison to high-fat diet</Title_Primary><Authors_Primary>Sampey,B.P.</Authors_Primary><Authors_Primary>Vanhoose,A.M.</Authors_Primary><Authors_Primary>Winfield,H.M.</Authors_Primary><Authors_Primary>Freemerman,A.J.</Authors_Primary><Authors_Primary>Muehlbauer,M.J.</Authors_Primary><Authors_Primary>Fueger,P.T.</Authors_Primary><Authors_Primary>Newgard,C.B.</Authors_Primary><Authors_Primary>Makowski,L.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>diet</Keywords><Keywords>model</Keywords><Keywords>human</Keywords><Keywords>metabolic</Keywords><Keywords>metabolic syndrome</Keywords><Keywords>syndrome</Keywords><Keywords>liver</Keywords><Keywords>ADIPOSE</Keywords><Keywords>adipose tissue</Keywords><Keywords>ADIPOSE-TISSUE</Keywords><Keywords>TISSUE</Keywords><Keywords>inflammation</Keywords><Keywords>HIGH-FAT</Keywords><Keywords>high-fat diet</Keywords><Reprint>Not in File</Reprint><Periodical>Obesity</Periodical><Volume>doi:10.1038/oby.2011.18</Volume><ZZ_JournalFull><f name="System">Obesity</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(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 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 1) and indicated which metabolites were responsible for the separation, with statistical significance (Tables 6 and 7). 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 long-chain acylcarnitines. The carnitine ester profiles (tetradecanoylcarnitine 14:0; palmitoylcarnitine 16:0; stearoylcarnitine 18:0 and linoleylcarnitine 18:2) tended to be higher in the CD group than in the SD. 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  ADDIN REFMGR.CITE <Refman><Cite><Author>Dayamand</Author><Year>2011</Year><RecNum>32396</RecNum><IDText>Carnitine: A novel health factor. An overview</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32396</Ref_ID><Title_Primary>Carnitine: A novel health factor. An overview</Title_Primary><Authors_Primary>Dayamand,C.D.</Authors_Primary><Authors_Primary>Krishmamurthy,N.</Authors_Primary><Authors_Primary>Ashakiran,S.</Authors_Primary><Authors_Primary>Shashidhar,K.N.</Authors_Primary><Date_Primary>2011</Date_Primary><Keywords>carnitine</Keywords><Keywords>health</Keywords><Keywords>factor</Keywords><Reprint>In File</Reprint><Start_Page>79</Start_Page><End_Page>89</End_Page><Periodical>Int.J.Pharm.Biomed.Res.</Periodical><Volume>2</Volume><ZZ_JournalStdAbbrev><f name="System">Int.J.Pharm.Biomed.Res.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(34). Interestingly, our findings fit to the one mass spectrometry based metabolic profiling reported by Koves and co-authors  ADDIN REFMGR.CITE <Refman><Cite><Author>Koves</Author><Year>2008</Year><RecNum>32397</RecNum><IDText>Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32397</Ref_ID><Title_Primary>Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance</Title_Primary><Authors_Primary>Koves,T.R.</Authors_Primary><Authors_Primary>Ussher,J.R.</Authors_Primary><Authors_Primary>Noland,R.C.</Authors_Primary><Authors_Primary>Slent&lt;,D.</Authors_Primary><Authors_Primary>Mosedale,M.</Authors_Primary><Authors_Primary>Ilkayeva,O.</Authors_Primary><Authors_Primary>Bain,J.</Authors_Primary><Authors_Primary>Stevens,R.</Authors_Primary><Authors_Primary>Dyck,J.R.</Authors_Primary><Authors_Primary>Negard,C.B.</Authors_Primary><Authors_Primary>Lopaschuk,G.D.</Authors_Primary><Authors_Primary>Muoio,D.M.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>mitochondrial</Keywords><Keywords>fatty acid</Keywords><Keywords>Fatty acid oxidation</Keywords><Keywords>acid</Keywords><Keywords>Oxidation</Keywords><Keywords>Skeletal muscle</Keywords><Keywords>muscle</Keywords><Keywords>muscle insulin resistance</Keywords><Keywords>insulin</Keywords><Keywords>insulin resistance</Keywords><Keywords>Insulin-resistance</Keywords><Keywords>resistance</Keywords><Reprint>In File</Reprint><Start_Page>45</Start_Page><End_Page>56</End_Page><Periodical>Cell Metab.</Periodical><Volume>7</Volume><ZZ_JournalStdAbbrev><f name="System">Cell Metab.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(35) showing that long-chain acylcarnitines (16:0,18:0, 18:1 and 18:2) 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  ADDIN REFMGR.CITE <Refman><Cite><Author>Mihalik</Author><Year>2010</Year><RecNum>32398</RecNum><IDText>Increased levels of plasma acylcarnitines in obesity and type 2 diabetes and identification of a marker of glucolipotoxicity</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32398</Ref_ID><Title_Primary>Increased levels of plasma acylcarnitines in obesity and type 2 diabetes and identification of a marker of glucolipotoxicity</Title_Primary><Authors_Primary>Mihalik,S.J.</Authors_Primary><Authors_Primary>Goodpaster,B.H.</Authors_Primary><Authors_Primary>Kelley,D.E.</Authors_Primary><Authors_Primary>Chace,D.H.</Authors_Primary><Authors_Primary>Vockley,J.</Authors_Primary><Authors_Primary>Toledo,F.G.</Authors_Primary><Authors_Primary>Delany,J.P.</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>Plasma</Keywords><Keywords>Acylcarnitines</Keywords><Keywords>acylcarnitine</Keywords><Keywords>obesity</Keywords><Keywords>Type 2 Diabetes</Keywords><Keywords>diabetes</Keywords><Keywords>marker</Keywords><Reprint>In File</Reprint><Start_Page>1695</Start_Page><End_Page>1700</End_Page><Periodical>Obesity</Periodical><Volume>18</Volume><ZZ_JournalFull><f name="System">Obesity</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(36). 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 by Adams et al. for type 2 diabetic women  ADDIN REFMGR.CITE <Refman><Cite><Author>Adams</Author><Year>2009</Year><RecNum>32399</RecNum><IDText>Plasma acylcarnitine profiles suggest incomplete long-chain fatty acid beta-oxidation and altered tricarboxylic acid cycle activity in type 2 diabetic african-american women</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>32399</Ref_ID><Title_Primary>Plasma acylcarnitine profiles suggest incomplete long-chain fatty acid beta-oxidation and altered tricarboxylic acid cycle activity in type 2 diabetic african-american women</Title_Primary><Authors_Primary>Adams,S.H.</Authors_Primary><Authors_Primary>Hoppel,C.I.</Authors_Primary><Authors_Primary>Lok,K.H.</Authors_Primary><Authors_Primary>Zhao,I.</Authors_Primary><Authors_Primary>Wong,S.W.</Authors_Primary><Authors_Primary>Minkler,P.E.</Authors_Primary><Authors_Primary>Hwang,D.H.</Authors_Primary><Authors_Primary>Newman,J.W.</Authors_Primary><Authors_Primary>Garvey,W.T.</Authors_Primary><Date_Primary>2009</Date_Primary><Keywords>Plasma</Keywords><Keywords>acylcarnitine</Keywords><Keywords>fatty acid</Keywords><Keywords>acid</Keywords><Keywords>beta-oxidation</Keywords><Keywords>tricarboxylic acid cycle</Keywords><Keywords>Activity</Keywords><Keywords>diabetic</Keywords><Keywords>African-American</Keywords><Keywords>African American</Keywords><Keywords>women</Keywords><Reprint>In File</Reprint><Start_Page>1073</Start_Page><End_Page>1081</End_Page><Periodical>J.Nutr.</Periodical><Volume>139</Volume><ZZ_JournalFull><f name="System">Journal of Nutrition</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Nutr.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(37). 
A specific accumulation of saturated acylcarnitines was found in plasma of rats fed the cafeteria diet, indicating inefficient beta-oxidation  ADDIN REFMGR.CITE <Refman><Cite><Author>Makowski</Author><Year>2009</Year><RecNum>6208</RecNum><IDText>Metabolic profiling of PPARalpha-/- mice reveals defetcts in carnitine and amino acid homeostasis that are partially reversed by oral carnitine supplementation</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>6208</Ref_ID><Title_Primary>Metabolic profiling of PPARalpha-/- mice reveals defetcts in carnitine and amino acid homeostasis that are partially reversed by oral carnitine supplementation</Title_Primary><Authors_Primary>Makowski,L.</Authors_Primary><Authors_Primary>Noland,R.C.</Authors_Primary><Authors_Primary>Koves,T.R.</Authors_Primary><Authors_Primary>Xing,W.</Authors_Primary><Authors_Primary>Ilkayeva,O.R.</Authors_Primary><Authors_Primary>et.al</Authors_Primary><Date_Primary>2009</Date_Primary><Keywords>profiling</Keywords><Keywords>mice</Keywords><Keywords>carnitine</Keywords><Keywords>amino acid</Keywords><Keywords>acid</Keywords><Keywords>homeostasis</Keywords><Keywords>oral</Keywords><Keywords>supplementation</Keywords><Reprint>Not in File</Reprint><Start_Page>586</Start_Page><End_Page>604</End_Page><Periodical>FASEB J.</Periodical><Volume>23</Volume><Web_URL_Link4><f name="System">FASEB Journal</f></Web_URL_Link4><ZZ_JournalStdAbbrev><f name="System">FASEB J.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Koves</Author><Year>2008</Year><RecNum>6209</RecNum><IDText>Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle resistance</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>6209</Ref_ID><Title_Primary>Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle resistance</Title_Primary><Authors_Primary>Koves,T.R.</Authors_Primary><Authors_Primary>Ussher,J.R.</Authors_Primary><Authors_Primary>Noland,R.C.</Authors_Primary><Authors_Primary>Slentz,D.</Authors_Primary><Authors_Primary>Mosedale,M.J.</Authors_Primary><Authors_Primary>et al.</Authors_Primary><Date_Primary>2008</Date_Primary><Keywords>fatty acid</Keywords><Keywords>fatty acid oxidation</Keywords><Keywords>acid</Keywords><Keywords>oxidation</Keywords><Keywords>skeletal muscle</Keywords><Keywords>muscle</Keywords><Keywords>resistance</Keywords><Reprint>Not in File</Reprint><Start_Page>45</Start_Page><End_Page>56</End_Page><Periodical>Cell Metab.</Periodical><Volume>7</Volume><ZZ_JournalStdAbbrev><f name="System">Cell Metab.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(38,39), 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. The main group of identified glycerophospholipids was the lysoPC with the shorter chain fatty acids (14:0) and the lysoPE (20:0), which showed up-regulation in rats fed the cafeteria diet, whereas those with the long chain fatty acids, mainly polyunsaturated, were down-regulated in these same rats (i.e. the CD group) (table 6). 
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  ADDIN REFMGR.CITE <Refman><Cite><Author>Barber</Author><Year>2012</Year><RecNum>571</RecNum><IDText>Plasma lysophosphatidylcholine levels are reduced in obesity and type 2 diabetes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>571</Ref_ID><Title_Primary>Plasma lysophosphatidylcholine levels are reduced in obesity and type 2 diabetes</Title_Primary><Authors_Primary>Barber,M.N.</Authors_Primary><Authors_Primary>Risis,S.</Authors_Primary><Authors_Primary>Yang,C.</Authors_Primary><Authors_Primary>Meikle,P.J.</Authors_Primary><Authors_Primary>Staples,M.</Authors_Primary><Authors_Primary>Febbraio,M.A.</Authors_Primary><Authors_Primary>Bruce,C.R.</Authors_Primary><Date_Primary>2012</Date_Primary><Keywords>plasma</Keywords><Keywords>obesity</Keywords><Keywords>type 2 diabetes</Keywords><Keywords>diabetes</Keywords><Reprint>In File</Reprint><Start_Page>e41456</Start_Page><End_Page>doi: 10.1371/journal.pone.0041456</End_Page><Periodical>PloS One</Periodical><Volume>7 (7)</Volume><ZZ_JournalFull><f name="System">PloS One</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(40) and in human obese subjects  ADDIN REFMGR.CITE <Refman><Cite><Author>Zhao</Author><Year>2010</Year><RecNum>572</RecNum><IDText>Metabolomic fingerprints of fasting plasma and spot urine reveal human pre-diabetic metabolic traits</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>572</Ref_ID><Title_Primary>Metabolomic fingerprints of fasting plasma and spot urine reveal human pre-diabetic metabolic traits</Title_Primary><Authors_Primary>Zhao,X.</Authors_Primary><Authors_Primary>Fristche,J.</Authors_Primary><Authors_Primary>Wang,J.</Authors_Primary><Authors_Primary>Chen,J.</Authors_Primary><Authors_Primary>Ritting,K.</Authors_Primary><Authors_Primary>Schmitt-Kopplin,P.</Authors_Primary><Authors_Primary>Frische,A.</Authors_Primary><Authors_Primary>H&#xE4;ring,H.U.</Authors_Primary><Authors_Primary>Schleicher,E.D.</Authors_Primary><Authors_Primary>Xu,G.</Authors_Primary><Authors_Primary>Lehmann,R.</Authors_Primary><Date_Primary>2010</Date_Primary><Keywords>fingerprints</Keywords><Keywords>fasting</Keywords><Keywords>plasma</Keywords><Keywords>urine</Keywords><Keywords>human</Keywords><Keywords>metabolic traits</Keywords><Reprint>In File</Reprint><Start_Page>362</Start_Page><End_Page>374</End_Page><Periodical>Metabolomics</Periodical><Volume>6</Volume><ZZ_JournalFull><f name="System">Metabolomics</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(41). 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  ADDIN REFMGR.CITE <Refman><Cite><Author>Ha</Author><Year>2012</Year><RecNum>566</RecNum><IDText>The association of specific metabolites of lipid metabolism with markers of oxidative stress, inflammation and arterial stiffness in men with newly diagnosed type 2 diabetes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>566</Ref_ID><Title_Primary>The association of specific metabolites of lipid metabolism with markers of oxidative stress, inflammation and arterial stiffness in men with newly diagnosed type 2 diabetes</Title_Primary><Authors_Primary>Ha,C.Y.</Authors_Primary><Authors_Primary>Kim,J.K.</Authors_Primary><Authors_Primary>Paik,J.K.</Authors_Primary><Authors_Primary>Kim,O.Y.</Authors_Primary><Authors_Primary>Paik,Y.H.</Authors_Primary><Authors_Primary>Lee,E.J.</Authors_Primary><Authors_Primary>Lee,J.H.</Authors_Primary><Date_Primary>2012</Date_Primary><Keywords>lipid</Keywords><Keywords>lipid metabolism</Keywords><Keywords>metabolism</Keywords><Keywords>oxidative stress</Keywords><Keywords>inflammation</Keywords><Keywords>arterial stiffness</Keywords><Keywords>type 2 diabetes</Keywords><Keywords>diabetes</Keywords><Reprint>In File</Reprint><Start_Page>674</Start_Page><End_Page>682</End_Page><Periodical>Clin.Endocrinol.</Periodical><Volume>76</Volume><ZZ_JournalStdAbbrev><f name="System">Clin.Endocrinol.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(42). 
Furthermore, recently lysoPC has also been shown to have a role in the metabolism of glucose. Yea et al. 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  ADDIN REFMGR.CITE <Refman><Cite><Author>Yea</Author><Year>2009</Year><RecNum>573</RecNum><IDText>Lysophosphatidylcholine activates adipocyte glucose uptake and lowers blood glucose levels in murine models of diabetes</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>573</Ref_ID><Title_Primary>Lysophosphatidylcholine activates adipocyte glucose uptake and lowers blood glucose levels in murine models of diabetes</Title_Primary><Authors_Primary>Yea,K.</Authors_Primary><Authors_Primary>Kim,J.</Authors_Primary><Authors_Primary>Yoon,J.H.</Authors_Primary><Authors_Primary>Kwon,T.</Authors_Primary><Authors_Primary>Kim,J.H.</Authors_Primary><Authors_Primary>Lee,B.D.</Authors_Primary><Authors_Primary>Lee,H.J.</Authors_Primary><Authors_Primary>Kim,J.L.</Authors_Primary><Authors_Primary>Lee,T.G.</Authors_Primary><Authors_Primary>Baek,M.C.</Authors_Primary><Authors_Primary>Park,H.S.</Authors_Primary><Authors_Primary>Ohba,M.</Authors_Primary><Authors_Primary>Suh,P.G.</Authors_Primary><Authors_Primary>Ryu,S.H.</Authors_Primary><Date_Primary>2009</Date_Primary><Keywords>adipocyte</Keywords><Keywords>glucose</Keywords><Keywords>glucose uptake</Keywords><Keywords>models</Keywords><Keywords>diabetes</Keywords><Reprint>In File</Reprint><Start_Page>33833</Start_Page><End_Page>33840</End_Page><Periodical>J.Biol.Chem.</Periodical><Volume>284</Volume><ZZ_JournalFull><f name="System">Journal of Biological Chemistry</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Biol.Chem.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(43) 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 lysophosphatidyl-ethanolamines (lysoPE) is not as wide but, it seems that they can be synthesized in a similar way to lysoPC  ADDIN REFMGR.CITE <Refman><Cite><Author>Makide</Author><Year>2009</Year><RecNum>575</RecNum><IDText>Emerging lysophospholipid mediators, lysophosphatydilserine, lysophosphatydilethanolamine, and lysophosphatydilglycerol</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>575</Ref_ID><Title_Primary>Emerging lysophospholipid mediators, lysophosphatydilserine, lysophosphatydilethanolamine, and lysophosphatydilglycerol</Title_Primary><Authors_Primary>Makide,K.</Authors_Primary><Authors_Primary>Kitamura,H.</Authors_Primary><Authors_Primary>Sato,Y.</Authors_Primary><Authors_Primary>Okutani,M.</Authors_Primary><Authors_Primary>Aoki,J.</Authors_Primary><Date_Primary>2009</Date_Primary><Reprint>In File</Reprint><Start_Page>135</Start_Page><End_Page>139</End_Page><Periodical>Prostaglandin and other lipid mediators</Periodical><Volume>89</Volume><ZZ_JournalFull><f name="System">Prostaglandin and other lipid mediators</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(44). In agreement with this view, our study shows that changes in lysoPE levels in CD and SD rats were similar, being up-regulated in the case of those with saturated fatty acids and down-regulated in those with unsaturated ones.
The addition of Rubus extract to the CD produced some interesting results. The  supplement decreased 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 could be related to the low LPL activity found in these animals when compared to those fed the CD, consistent with the known effect of this enzyme controlling the clearance of circulating TAG  ADDIN REFMGR.CITE <Refman><Cite><Author>Foubert</Author><Year>1996</Year><RecNum>17447</RecNum><IDText>Lipoprotein lipase:  A multifunctional enzyme in lipoprotein metabolism</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>17447</Ref_ID><Title_Primary>Lipoprotein lipase:  A multifunctional enzyme in lipoprotein metabolism</Title_Primary><Authors_Primary>Foubert,L.</Authors_Primary><Authors_Primary>Benlian,P.</Authors_Primary><Authors_Primary>Turpin,G.</Authors_Primary><Date_Primary>1996</Date_Primary><Keywords>lipoprotein</Keywords><Keywords>lipoprotein lipase</Keywords><Keywords>lipase</Keywords><Keywords>enzyme</Keywords><Keywords>lipoprotein metabolism</Keywords><Keywords>metabolism</Keywords><Keywords>lpl</Keywords><Keywords>hydrolysis</Keywords><Keywords>triglycerides</Keywords><Keywords>function</Keywords><Keywords>lipoprotein lipase deficiency</Keywords><Keywords>lipase deficiency</Keywords><Keywords>deficiency</Keywords><Keywords>chylomicronemia</Keywords><Keywords>general population</Keywords><Keywords>Population</Keywords><Keywords>autosomal recessive</Keywords><Keywords>Lipoprotein lipase gene</Keywords><Keywords>gene</Keywords><Keywords>gene mutations</Keywords><Keywords>mutations</Keywords><Keywords>fasting</Keywords><Keywords>Plasma</Keywords><Keywords>plasma triglyceride</Keywords><Keywords>Triglyceride</Keywords><Keywords>triglyceride levels</Keywords><Keywords>acute</Keywords><Keywords>acute pancreatitis</Keywords><Keywords>pancreatitis</Keywords><Keywords>lipid</Keywords><Keywords>lipid profile</Keywords><Reprint>Not in File</Reprint><Start_Page>207</Start_Page><End_Page>210</End_Page><Periodical>Presse Med.</Periodical><Volume>25</Volume><ZZ_JournalStdAbbrev><f name="System">Presse Med.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite><Cite><Author>Merkel</Author><Year>2002</Year><RecNum>MERKEL2002A</RecNum><IDText>Lipoprotein lipase: genetics, lipid uptake, and regulation</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>MERKEL2002A</Ref_ID><Title_Primary>Lipoprotein lipase: genetics, lipid uptake, and regulation</Title_Primary><Authors_Primary>Merkel,M.</Authors_Primary><Authors_Primary>Eckel,R.H.</Authors_Primary><Authors_Primary>Goldberg,I.J.</Authors_Primary><Date_Primary>2002/12</Date_Primary><Keywords>triglycerides</Keywords><Keywords>chylomicronemia</Keywords><Keywords>atherosclerosis</Keywords><Keywords>lipoproteins</Keywords><Keywords>lipid metabolism</Keywords><Keywords>mutations</Keywords><Keywords>human</Keywords><Keywords>promoter</Keywords><Keywords>review</Keywords><Keywords>lipoprotein</Keywords><Keywords>lipoprotein lipase</Keywords><Keywords>lipase</Keywords><Keywords>genetics</Keywords><Keywords>lipid</Keywords><Keywords>uptake</Keywords><Keywords>regulation</Keywords><Keywords>LPL</Keywords><Keywords>plasma</Keywords><Keywords>triglyceride</Keywords><Keywords>HDL</Keywords><Keywords>LPL gene</Keywords><Keywords>gene</Keywords><Keywords>mutation</Keywords><Keywords>plasma lipids</Keywords><Keywords>plasma lipid</Keywords><Keywords>lipids</Keywords><Keywords>coronary</Keywords><Keywords>coronary heart disease</Keywords><Keywords>heart</Keywords><Keywords>heart disease</Keywords><Keywords>disease</Keywords><Keywords>liver</Keywords><Keywords>tissue</Keywords><Keywords>gene regulation</Keywords><Reprint>Not in File</Reprint><Start_Page>1997</Start_Page><End_Page>2006</End_Page><Periodical>J.Lipid Res.</Periodical><Volume>43</Volume><Issue>12</Issue><ZZ_JournalFull><f name="System">Journal of Lipid Research</f></ZZ_JournalFull><ZZ_JournalStdAbbrev><f name="System">J.Lipid Res.</f></ZZ_JournalStdAbbrev><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(45,46). The metabolomic profile revealed that the CDR and CD diets had opposite effects on lysoPE concentrations when compared to the animals on the SD. 
Therefore, 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 changes in the AUCs for insulin and glucose 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 have been standardized in terms of their contents of specific flavonols and anthocyanins, since the contents of these phytochemicals is known to fluctuate depending on environmental conditions  ADDIN REFMGR.CITE <Refman><Cite><Author>Ramos-Solano</Author><Year>2014</Year><RecNum>592</RecNum><IDText>Annual changes in bioactive contents and production in field-grown blackberry after inoculation with Pseudomonas fluorescens</IDText><MDL Ref_Type="Journal"><Ref_Type>Journal</Ref_Type><Ref_ID>592</Ref_ID><Title_Primary>Annual changes in bioactive contents and production in field-grown blackberry after inoculation with Pseudomonas fluorescens</Title_Primary><Authors_Primary>Ramos-Solano,B.</Authors_Primary><Authors_Primary>Garc&#xED;a-Villaraco,A.</Authors_Primary><Authors_Primary>Gutierrez-Ma&#xF1;ero,F.J.</Authors_Primary><Authors_Primary>Lucas,J.A.</Authors_Primary><Authors_Primary>Bonilla,A.</Authors_Primary><Authors_Primary>Garcia-Seco,D.</Authors_Primary><Date_Primary>2014</Date_Primary><Reprint>In File</Reprint><Start_Page>1</Start_Page><End_Page>8</End_Page><Periodical>Plant Physiology and Biochemistry</Periodical><Volume>74C DOI: 10.1016/j.plaphy.2013.10.029</Volume><ZZ_JournalFull><f name="System">Plant Physiology and Biochemistry</f></ZZ_JournalFull><ZZ_WorkformID>1</ZZ_WorkformID></MDL></Cite></Refman>(47). Furthermore absorption is subject to microfloral activity (9) so this also needs to be controlled. Alternatively, studies using more purified extracts may yield more information about the active compounds.
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 such 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 Fundaci�n Ram�n Areces (CIVP16A1835) of Spain. AGL2009-08324, BES-2010-038057. 
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Figure 1. 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) (�%).

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