Chronic intake of proanthocyanidins and docosahexaenoic acid improves skeletal muscle oxidative capacity in diet-obese rats Ester Casanova, Laura Baselga-Escudero, Aleix Ribas-Latre, L´ıdia Ced´o Anna Arola-Arnal, Montserrat Pinent, Cinta Blad´e, Llu´ıs Arola, M. Josepa Salvad´o PII: DOI: Reference:
S0955-2863(14)00113-2 doi: 10.1016/j.jnutbio.2014.05.003 JNB 7207
To appear in:
The Journal of Nutritional Biochemistry
Received date: Revised date: Accepted date:
13 January 2014 15 April 2014 2 May 2014
Please cite this article as: Casanova Ester, Baselga-Escudero Laura, Ribas-Latre Aleix, Arola-Arnal L´ıdia Ced´o Anna, Pinent Montserrat, Blad´e Cinta, Arola Llu´ıs, Josepa Salvad´o M, Chronic intake of proanthocyanidins and docosahexaenoic acid improves skeletal muscle oxidative capacity in diet-obese rats, The Journal of Nutritional Biochemistry (2014), doi: 10.1016/j.jnutbio.2014.05.003
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Chronic intake of proanthocyanidins and docosahexaenoic acid
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improves skeletal muscle oxidative capacity in diet-obese rats
Ester Casanova, Laura Baselga-Escudero, Aleix Ribas-Latre, Lídia Cedó Anna Arola-Arnal, Montserrat Pinent, Cinta Bladé, Lluís Arola, M. Josepa
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Salvadó*
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Grup de Nutrigenomica. Departament de Bioquimica i Biotecnologia. Universitat Rovira i Virgili. Campus Sescel·lades, 43007. Tarragona. Spain Corresponding Author: M. Josepa Salvadó
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Department of Biochemistry and Biotechnology,
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Univeristat Rovira i Virgili
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C/ Marcel·lí i Domingo s/n 43007 Tarragona
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Spain
Phone: +34977559567 Fax: +34977558232 E-mail: [email protected]
Running title: proanthocyanidins and omega3 improve muscle oxidation:
Keywords: obesity, docosahexaenoic acid, proanthocyanidins, skeletal muscle, mitochondria, β-oxidation
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ACCEPTED MANUSCRIPT Abbreviations AMPK (5’-AMP-activated protein kinase), Cafeteria diet (CD), CK (creatine
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kinase), COX (Cytrochrome c oxidase) ,CPT ( palmitoyl translocase) ,CS
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(citrate synthase), DHA (docosahexaenoic acid), EPA ( eicosapentanoic acid), ETC (electron transport chain), FA (fatty acid), FCCP (trifluorocarbonylcyanide
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phenylhydrazone), GSPE (grape seed proanthocyanidins extract, Mnsod (manganes superoxide), OXPHOS (oxidative phosphorylation), PPARs
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(peroxisome proliferator-activated receptors), PUFAs (polyunsaturated fatty acids), RQ (respiratory quotient), STD (standard diet),TCA ( tricarboxylic acid
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cycle).
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ACCEPTED MANUSCRIPT Abstract Obesity has become a worldwide epidemic. The cafeteria diet (CD) induces
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obesity and oxidative stress-associated insulin resistance. Polyunsaturated fatty
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acids and polyphenols are dietary compounds that are intensively studied as products that can reduce the health complications related to obesity. We
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evaluate the effects of 21 days of supplementation with grape seed
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proanthocyanidins extract (GSPE), docosahexaenoic-rich oil (DHA-OR) or both compounds (GSPE+DHA-OR) on skeletal muscle metabolism in diet-obese rats. The supplementation with different treatments did not reduce body weight although all groups used more fat as fuel, particularly when both products were
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co-administered; muscle β-oxidation was activated, the mitochondrial functionality and oxidative capacity was higher, and fatty acid uptake gene
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expressions were upregulated. In addition to these outcomes shared by all treatments, GSPE reduced insulin resistance, and improved muscle status.
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Both treatments increased 5’-AMP-activated protein kinase (AMPK)
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phosphorylation, which was consistent with higher plasma adiponectin levels. Moreover AMPK activation by DHA-OR was also correlated with an upregulation of peroxisome proliferator-activated receptor alpha (Pparα). GSPE+DHA-OR, in addition to activate AMPK and enhance fatty acid oxidation, increased the muscle gene expression of uncoupling protein 2 (Ucp2). In conclusion GSPE+DHA-OR induced modifications that improved muscle status and could counterbalance the deleterious effects of obesity, and such modifications are mediated at least in part, through the AMPK signaling pathway.
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ACCEPTED MANUSCRIPT Introduction Obesity is a complex, multifactorial disease characterized by an increase in
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body weight or, more specifically, an increase in adipose tissue. It is prevalent
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in both developed and developing countries and affects children as well as adults[1]. Obesity produces adverse health consequences, such as
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dyslipidemia, insulin resistance, hypertension, type 2 diabetes and
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cardiovascular disease. To reduce food intake and increase physical activity are the main strategies followed to prevent and reduce obesity. New strategies for weight control management contemplate the use of bioactive compounds in the diet providing potential antiobesity effects. In this sense, medium chain
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triglycerides increase energy expenditure and fatty acid oxidation [2,3], omega3 fatty acids reduce adiposity index [4], and polyphenols reduce adipose index,
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inflammation, and stimulate thermogenesis [5–7]. Skeletal muscle has an
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important role in the overall utilization and oxidation of fatty acids (FAs). Specifically, skeletal muscle manifests reduced reliance upon FA oxidation in
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obesity compared to skeletal muscle of lean individuals. Biochemical marker levels are consistent with these findings, i.e. Carnitine palmitoyl translocase (CPT) 1 expression is diminished in obesity, with reduced capacity for FA oxidation[8]. Obesity may also interfere with mitochondrial bioenergetics by affecting cellular respiratory functions and oxidative pathways. In addition, there is a chronic elevation of circulating FA levels, a reduction in the expression of genes involved in mitochondrial biogenesis, an increased production of reactive oxygen species, and an impaired mitochondrial biogenesis and function in skeletal muscle [9,10]. The cafeteria diet (CD) is a robust model of human metabolic syndrome compared to traditional lard-based high-fat diets [6]. In this 4
ACCEPTED MANUSCRIPT model, animals are fed with a standard diet (STD) and concurrently offered highly palatable, energy-dense foods ad libitum. This diet promotes voluntary
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hyperphagia [11] that results in rapid weight gain and increases fat pad mass
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and prediabetic parameters, such as glucose and insulin intolerance and dyslipidemia [12].
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The use of dietary compounds that reduce health complications related to these
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pathologies is emerging as a new approach. Previous reports have raised the possibility that polyphenols and omega-3 polyunsaturated fatty acids (PUFAs) are good dietary compounds for reducing obesity-induced metabolic syndrome [13–16]. Treatment of rodents with different proanthocyanidins results in
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activation of -oxidation [17,18], regulation of expression/activity of enzymes of lipogenesis and fatty acid oxidation[19], and upregulation of genes related to
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oxidative phosphorylation [17]. Likewise, omega-3 PUFAs are involved in a
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variety of mitochondrial processes. Particularly, docosahexaenoic acid (DHA) consumption, stimulated genes related to mitochondrial functionality, FA
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oxidation and inhibited genes involved in FA synthesis [20]. Moreover, they are natural ligands of PPARs, such as PPARα that is activated by several PUFAs [21,22], with eicosapentaenoic acid (EPA) and DHA as the predominantly active biological components [23,24]. A synergistic effect between polyphenols and omega-3 PUFAs has been shown [25]. In the present study, we considered the possibility that intake of grape seed proanthocyanidins extract (GSPE) and/or DHA-rich oil (DHA-OR) concomitant with a CD could influence muscle fat and carbohydrate oxidation rates, while improving the deleterious effects associated with obesity-induced metabolic syndrome. Our hypothesis is that the consumption of GSPE and 5
ACCEPTED MANUSCRIPT DHA-OR could increase mitochondrial oxidative capacity in skeletal muscle on obesity. Besides, AMPK activation activates fatty acid oxidation, insulin
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signaling and could be modulated by adipokines such as leptin and adiponectin,
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important regulators of whole-body energy metabolism [26,27] . Given the key role of AMPK on energy homeostasis, we hypothetized that the effects of the
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bioactive compounds could be though affecting hormonal control and through
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modulating the enzyme AMPK.
Thus, we evaluated the effect of individual or simultaneous administration of GSPE and DHA-OR by analyzing the respiratory quotient (RQ) ratio (using indirect calorimetry (IC)), insulin resistance, plasma adipokine levels,
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mitochondrial functionality (measuring oxygen consumption of isolated mitochondria from skeletal muscle), mitochondrial enzymatic activities and
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expression of key metabolic genes .
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Materials and Methods Grape seed proanthocyanidins extract GSPE was provided by Les Dérives Résiniques et Terpéniques (Dax, France). The GSPE composition used in this study has been previously analyzed [28]: catechin (58 µmol/g), epicatechin (52 µmol/g), epigallocatechin (5.50 µmol/g), epicatechin gallate (89 µmol/g), epigallocatechin gallate (1.40 µmol/g), dimeric procyanidins (250 µmol/g), trimeric procyanidins (1568 µmol/g), tetrameric procyanidins (8.8 µmol/g), pentameric procyanidins (0.73 µmol/g) and hexameric procyanidins (0.38 µmol/g).
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ACCEPTED MANUSCRIPT Oil rich in docosahexaenoic acid The DHA-OR was provided by Market DHATm-S (Columbia, MD, USA). The
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nutritional composition of the oil derived from marine alga, Schizochytrium sp., a
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rich source of DHA with sunflower lecithin anad rosemary extract (flavoring). The fatty acid profile of DHA-OR was 5.6% of 14:0, 16.1% of 16:0, 0.9% of
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18:0, 15% of 18:1n-9, 1.4% of 18:2n-6, 0.5% of 20:4n-6 (ARA), 1.1% of 20:5n-3
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(EPA), 16.2% of 22:5n-6 (DPA), 38.8% of 22:6n-3 (DHA) and 0.5% of others. The oil also contained tocopherols and ascorbyl palmitate as antioxidants to provide stability.
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Animals and diets
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Male Wistar rats weighing 150 g were supplied by Charles River Laboratories (Barcelona, Spain). The animals were housed in a 12 h light-dark-cycle at 21-
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23ºC. Following adaptation week, animals were divided into five equal groups (7
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rats/group) and housed individually to control the food intake. One group was the STD group, fed with standard chow diet (Panlab 04, Barcelona, Spain) ad libitum,and the remaining 4 groups were fed with STD plus CD consisting of hypercaloric diet with industrially processed foods designed for human consumption (cookies, cheese portions, bacon, foie-gras, sugary milk, cupcakes and carrots) composed of 35.20% carbohydrate, 23.40% fat, 11.70% protein, and 28.39 % water and 1.31% fiber. Animals were allowed free access to standard chow and water in addition to CD ad libitum. During the day, food was removed. Food consumption and weight gain were monitored weekly for 10 weeks until animals were 20% overweight compared with the STD group. Subsequently, different treatments were administered jointly with the CD for 21 7
ACCEPTED MANUSCRIPT days where food consumption and body wegith were monitored every 5 days. One group (CD+GSPE) was supplemented with 25 mg GSPE /kg body weight
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dissolved in 5% Arabic gum (Sigma-Aldrich, Madrid, Spain). The second group
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was supplemented with 500 mg oil-rich DHA (38.8%) / kg body weight dissolved in 5% of Arabic gum (CD+DHA-OR). The third group received GSPE (25 mg/kg
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body weight) plus 500 mg oil-rich DHA (38.8%)/ kg body weight dissolved in 5% of Arabic gum (CD+GSPE+DHA-OR)). All groups received the same volume of
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Arabic gum. The rats were treated in the afternoon, and then allowed free access to fresh portions of chow and/or CD during the night. On day 21 of treatment, all rats were fasted for 3-hours before being anesthetized using 50 mg/kg body weight of sodium pentobarbital (Fagron Iberica, Terrasa, Spain)
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and sacrificed by abdominal aorta exsanguination. Blood was collected using
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heparin (Deltalab, Barcelona, Spain). Plasma was obtained by centrifugation (1500 x g, 15 min, 4ºC) and stored at -80ºC until the subsequent plasma
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parameter analysis. Gastronecmius muscles were excised, weighed,
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immediately frozen in liquid N2 and stored at -80ºC until further analysis. Small portions of the gastrocnemius skeletal muscle were also placed on ice and used to isolate mitochondria for enzymatic assays and mitochondrial functionality. Mesenteric, perirrenal, and epydimidal adipose tissues were excised and weighed. All procedures were approved by the Animal Ethics Committee of our university.
Plasmatic Parameters Creatine Kinase (CK) and Glucose were measured using enzymatic colorimetric kits (QCA, Barcelona, Spain). Insulin was assayed by ELISA method following 8
ACCEPTED MANUSCRIPT the manufacturer’s instructions (Mercodia, Uppsala, Sweden). The HOMA index was calculated using the fasting values of glucose and insulin with the following
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formula:
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Adiponectin and Leptin levels were quantified using specific EIAs according to
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the manufacturer’s instructions (Biosource International, Inc, USA).
Indirect calorimetry
Assessment of respiratory metabolism and dates of analysis were performed in
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a ventilated hood system (Panlab Harvard Apparatus, Barcelona, Spain). RQ
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values (VO2 / VCO2) were recorded by IC measurements at 10 and 20 days of
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treatment during 6-hours of the postprandial period (9.00 h – 15.00 h).
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Mitochondrial respiration by high-resolution tracking The piece of gastrocnemius muscle placed in ice-cold homogenization medium buffer (100 mM sucrose, 50 mM KCl, 20 mM K+-TES, 1 mM EDTA and 0.2% (w/w) FA free-BSA (Sigma-Aldrich, Madrid, Spain) was used for mitochondrial respiratory assays. Mitochondria used for respiratory assays were isolated by differential centrifugations as described by Hoeks, J. [29]. Mitochondrial functionality was recorded at 37ºC, under stirring at 350 rpm, by a polarographic oxygen sensor in a two-chamber Oxygraph (OROBOROS® Instruments, Innsbruck, Austria). The oxygen flux was expressed in nmol O 2 per mg mitochondrial protein per minute. 9
ACCEPTED MANUSCRIPT To view the bioenergetics in freshly prepared mitochondria, we modulated oxidative phosphorylation (OXPHOS) function in the following ways: basal state
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(State2), the OXPHOS coupling effect (State3) stimulated with +450 µM ADP
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(Sigma-Aldrich, Madrid, Spain), non-phosphorylating respiration (State4) with the inhibition of ATP synthesis by the addition of 1 µg/ml oligomycin, and
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OXPHOS non-coupling effect (StateU) with +0.5 µM carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich, Madrid, Spain)
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addition to obtain the maximal respiratory capacity. The measurement of the OXPHOS system and the operation of the tricarboxylic acid cycle (TCA) were carried out using 3 different sets of substrates: a) 5 mM pyruvate +2.5 mM malate (glycolytic substrates) to show the functionality of the pyruvate
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dehydrogenase and TCA functionality; b)10 mM glutamate (complex I
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substrate) +10 mM succinate (complex II substrate) amino acids substrates to activate mitochondria respiration were used in combination of 0.5 µM of
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rotenone to inhibit complex I in order to study complex II capacity alone; c) 2
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mM L-carnitine +50 µM palmitoyl-CoA (FA substrate to induce β-oxidation). Briefly, 0.2 mg of protein where used by pyruvate-malate and glutamate-malate -succinate sets; by the other hand 0.5 mg of protein were used in carnitine+palmitoyl-CoA set and each set incubated in 2 ml respiration buffer.
Mitochondrial enzymatic activity Pieces of gastrocnemius were weighed and placed on ice for subsequent mitochondrial fraction isolation to analyze cytochrome c oxidase (COX), citrate synthase (CS) and ATPase activities according to the methods of Fuster et
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ACCEPTED MANUSCRIPT al.[30], Srere [31] and Bergmeyer, respectively, as we have previously
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detailed[32].
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RNA extraction and quantitative real-time PCR (qRT-PCR) analysis Total RNA was isolated from the gastrocnemius skeletal muscle tissue as
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detailed previously by Casanova et al.[33]. Specific Taqman Assay-on-Demand
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Probes were used: Peroxisome proliferator activated receptor alpha (Pparα) (Rn00566193_m1), Carnitine palmitoyltransferase 1b (Cpt1b) (Rn005664242_m1), Uncoupling protein 3 (Ucp3) (Rn00565874_m1), Uncoupling protein 2 (Ucp2) (Rn00571166_m1), ATPsynthase (Atpase)
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(Rn01527025_m1), Manganese Superoxide dismutase (Mnsod) (Rn00566942_g1), Lipoprotein Lipase (Lpl) (Rn00561482_m1), FA translocator
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CD36 (Cd36) (Rn00580728_m1). Cyclophilin peptidylprolyl isomerase A (Ppia)
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was used as an endogenous control gene (Rn00690933_m1). The relative
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mRNA expression levels were calculated using the 2-∆∆Ct method.
Western Blot (WB) analyses Protein was extracted from the frozen gastrocnemius skeletal muscle in RIPA lysis buffer (150 mM Tris-HCl, 1% Triton x-100, SDS 0.1%, 167 mM NaCl, 0.5% Na-deoxycholate) with a protease inhibitor cocktail 1/1000 (Sigma –Aldrich St. Louis, MO, USA), PMSF 1/100 (FLUKA-Biochemika, Switzerland) and a phosphatase inhibitor cocktail: cocktail II 1/100, cocktail III 1/100 (SigmaAldrich St.Louis, MO, USA). Total protein levels were determined using the Bradford method[34]. Proteins were loaded and run on a 10% SDSpolyacrylamide gel. Samples were transferred to PVDF membrane (Bio-Rad, 11
ACCEPTED MANUSCRIPT Hercules, CA, USA) using a transblot apparatus (Bio-Rad, Hercules, CA, USA) and subsequently blocked at room temperature with 5% (wt/vol) non-fat milk in
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TBS-T buffer (Tris-Buffered saline, 0.5% Tween-20). Primary antibodies were
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incubated overnight with shaking at 4ºC: rabbit AMPKα primary antibody, phospho AMPK α primary antibody (Cell Signaling Technology, Beverly, MA,
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USA), or rabbit α-tubulin (Sigma- Aldrich St. Louis, MO, USA). Blots were washed in TBST buffer and incubated with peroxidase-conjugated anti-rabbit
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secondary antibody (GE Healthcare, Buckinghamshire, UK) 1-hour. . Immunoreactive proteins were visualized using chemiluminiscence substrate kit ECL PLUS (GE Healthcare Buckinghamshire, UK) according to manufacturer’s instructions. Digital images were taken with a GBOX Chemi XL 1.4 image
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system (Syngene Ozyme, France). Quantification content was carried out in
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Image J Software (NIH, USA). The results were expressed as the relative
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intensity of p-AMPK/AMPK.
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Statistical Analysis
The results are expressed as the mean ± SEM of seven animals. SPSS Statistics version 19 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Significant differences were analyzed by one-way ANOVA followed by the Tukey post-hoc test. A p-value ≤ 0.05 was considered statistically significant.
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ACCEPTED MANUSCRIPT Results Food intake changed significantly only at specific points along the 21
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days of treatment
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CD significantly increased body weight as compared to STD at 2 weeks. The difference was more pronounced at 10 weeks (STD: 439.14±10.35a; CD:
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518.50±6.97b). Moreover, RQ values were significantly lower in CD than in STD
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group (STD: 1.00±0.01a; CD: 0.88±0.01b), which indicated a preference for lipid oxidation. Table 1 shows that mesenteric, perirenal and epididymal adipose tissue weights were higher in CD groups, than in STD group. Figure 1A shows body weight measurements during the treatment with no
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differences between groups. Figure 1B shows the food intake of each group during the entire treatment. At day 5 of treatment, the CD+GSPE+DHA-OR
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group increased their food intake significantly compared to the control CD
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group. Conversely, after 21 days, food intake was significantly decreased in the
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CD+GSPE group compared to CD group.
GSPE+DHA-OR administration along with CD decreased RQ values At 10 days of treatment (Figure 2A), RQ values from the CD group were significantly higher throughout the postprandial period compared to the treated groups. In contrast, the CD+GSPE+DHA-OR group had significantly lower RQ values and were also lower than those of the STD group. Figure 2B shows that the differences in RQ values between the CD groups are reduced at 20 days of treatment. The RQ was significantly lower in the CD+GSPE+DHA-OR group
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ACCEPTED MANUSCRIPT than in the remaining groups only at the end of the measurements (postprandial
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5-6-hours).
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GSPE and DHA-OR administration increased adiponectin levels
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Plasma adiponectin levels were significantly higher in CD+GSPE and CD+DHAOR groups compared to the CD group, while no significant differences were
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found in other treatment groups (Figure 3A). Moreover, leptin levels (Figure 3B) were significantly increased after CD+GSPE+DHA-OR administration compared
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to the other treatments.
GSPE administration reversed insulin resistance induced by CD
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Glucose plasma values did not differ between the CD and treated groups
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although all of them were increased compared to the values for the STD group (Figure 4A). Plasma insulin was decreased significantly in the CD+GSPE group,
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approaching the STD group values (Figure4B). The HOMA index (Figure 4C) was significantly decreased in the CD+GSPE group compared to the CD+DHAOR group, and was similar to STD group values.
GSPE and DHA-OR administration along with CD improved mitochondrial respiration compared to the CD group. The results of assessing the functionality of the OXPHOS system are described in Table 2. Animals fed with CD+DHA-OR had higher rates of respiration in all states compared to the other groups using three different sets of substrates in
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ACCEPTED MANUSCRIPT an aim to show the electron transport chain functionality between different sources of nutrients, especially in State3 which increased ATP production and
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in StateU that represents the respiratory maximum capacity. Mitochondrial
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respiration was lower in Palmitoyl-CoA oxidation set than in the other two substrate sets in the CD group. Moreover mitochondrial respiration in Palmitoyl-
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an increase of StateU by all treated groups.
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CoA oxidation set was increased in CD+GSPE and CD+DHA-OR groups with
Both GSPE and DHA-OR administration affected the expression of genes related to FA oxidation in skeletal muscle.
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Figure 5 shows that CD+DHA-OR treatment significantly increased Pparα, Cd36, Lpl and Cpt1b expression in muscle compared to the CD group.
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Additionally, CD+GSPE treatment significantly increased Cd36, Lpl and Cpt1b
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gene expression. For the CD+GSPE+DHA-OR group, Ucp2, Lpl and Cpt1b
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were significantly overexpressed. It is important to note that Ucp3 mRNA levels in rats fed a CD were higher than in those on the STD diet, although there were no differences between treated groups. None of the treatments altered ATPase or Mnsod gene expression.
GSPE administration increased ATPase activity, while GSPE and DHA-OR coadministration increased CS activity. COX activity showed no difference between groups. However, ATPase activity was higher in the CD+GSPE group compared to the other groups. CS activity in
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ACCEPTED MANUSCRIPT the CD+GSPE+DHA group was significantly elevated compared to the CD
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GSPE administration decreased CK plasma values
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group (Table3).
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CK, a plasma indicator of muscle damage, was significantly decreased by CD+GSPE administration with respect to the CD group, and also when
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administered concomitantly with DHA-OR. CK levels in both groups appeared to be restored to levels comparable to STD group (Table 3).
in skeletal muscle.
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AMPK phosphorylation was activated by GSPE or/and DHA-OR treatments
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Figure 6 shows that the administration of either GSPE or DHA-OR activated
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AMPK phosphorylation and significantly increased the ratio of
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phosphorylated/unphosphorylated AMPK compared to CD.
Discussion
Due to its high palatability, CD allows rodents to become hyperphagic by reducing their control over their food intake [11]. As a result of 10 weeks of eating the hypercaloric CD ad libitum, Wistar rats become obese, showing a 20% increase in body weight with higher mesenteric, perirenal and epididymal adipose tissue weights; with an adiposity index approximately twice STD-fed rat. Furthermore, CD rats are hypertriglyceridemic, hypercholesterolemic, hyperinsulinemic, and hyperleptinemic, with a higher HOMA index than STD
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ACCEPTED MANUSCRIPT rats[35–37]. After 10 weeks of consuming CD, GSPE, DHA-OR or a combination of both were added to the CD for 21 days. None of these
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treatments significantly improved body weight nor leptinemia, but increased
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plasma adiponectin. It is well known that obesity is associated to high concentrations of plasma leptin [38], and in the current study, GSPE did not
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suppress the elevated levels of leptin induced by obesity as found in another study where polyphenols suppressed the increases in leptin levels in high-fat-
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fed mice[26]. The addition of GSPE to the CD lowered insulinemia and HOMA index, thus improving the insulin resistance shown by CD rats in addition to their decreased TGs plasma levels observed in previous studies [33,35,39,40]. Published data showed that phenolic compounds reduced hyperinsulinemia by
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increasing adiponectin secretion and expression in obese diabetic mice [41,42].
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Here, we observed that adiponectin, not only reduced hyperinsulinemia but also stimulated β -oxidation in skeletal muscle via AMPK . The increase in FA
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oxidation following CD+GSPE treatment also modulate mitochondrial function
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substantiated by the high-resolution respirometry outcomes. Higher oxygen consumption in State2 and State4 of the palmitoyl-CoA oxidation demonstrated the increase of FA oxidation rates in ex vivo isolated mitochondria . Moreover, there was a high facility to increase, StateU, the maximum respiratory capacity and adapt OXPHOS system in extreme conditions by pyruvate/malate and palmitoyl-CoA pathways in CD+GSPE treated rats. Likewise, Pajuelo et al. demonstrated that GSPE modulates mitochondrial function, by adapting the OXPHOS system and increasing FA oxidation, also in brown adipose tissue of CD-fed rats [32].
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ACCEPTED MANUSCRIPT In the case of CD+GSPE rats, the significantly elevated transcript levels of Lpl, Cd36, Cpt1b and higher ATPase activity in skeletal muscle, concomitant with
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the reduced TG levels in plasma, was in agreement with the role of GSPE in
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redirecting circulating TGs to skeletal muscle for fat oxidation and ATP synthesis [33].In addition, in a study with a similar obesity model in rats, GSPE
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improved both lipidemia and the fat content in the liver [15]. All of these changes are consistent with high circulating adiponectin levels in the CD+GSPE
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group, because it has been reported that adiponectin increases the expression of molecules involved in FA transport (CD36) and combustion (acyl-coenzyme A oxidase) in skeletal muscle[38]. All these effects of GSPE underlie the
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prevention of muscle damage as shown by the lower plasma CK values. Likewise, DHA-OR increased plasma adiponectin levels, activated AMPK
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phopshorylation and elevated transcript levels of Lpl, Cd36, Cpt1b. In addition
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to increase β-oxidation in skeletal muscle, DHA-OR also increased mitochondrial OXPHOS functionality, with a maximum respiratory capacity
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shown in isolated mitochondria. In a previous study in which healthy rats were given an acute dose of DHA-OR plus a high fat overload, the results showed that DHA-OR facilitates the entry and uptake of long-chain FAs in skeletal muscle via Lpl and Cd36 overexpression, and increases the flux of FA oxidation to mitochondria by Cpt1b overexpression [33], as has been found in the present study with CD rats. This is likely to occur through upregulation of Pparα , because omega-3 PUFA are ligands of PPARα, resulting in a rapid upregulation of genes involved in lipid oxidation [43,44]; but may also be due to AMPK activation by increased adiponectin levels as other authors have reported [45–47]. 18
ACCEPTED MANUSCRIPT The effects of CD+GSPE+DHA-OR were similar to those of each separate compound in terms of muscle energy homeostasis and oxidative capacity. The
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increased mitochondrial oxidative capacity and fatty acid oxidation by GSPE
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and/or DHA-OR was reinforced by the results of RQ values at 10 days of treatment using a markedly higher percentage of fatty acid combustion over
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carbohydrates. Meanwhile, at the end of the treatment CD+GSPE+DHA-OR treated rats fuelled essentially FAs, in spite of a RQ ratio closer to the other
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groups, so the combination of both compounds caused the highest burning of fat overload. Again, GSPE+DHA-OR significantly upregulated gene expression of key markers of β-oxidation, Lpl and Cpt1b.These results agreed with the high functionality of mitochondria, and with the improvement of the unusual low
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oxygen consumption shown in CD group with palmitate as substrate. Once
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again, these effects occur through AMPK activation, which was concomitant with significantly elevated transcript levels of Ucp2. Pecqueur et al.[48] showed
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that UCP2 promotes mitochondrial FA oxidation while limiting mitochondrial
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catabolism of pyruvate, suggesting that the metabolic and proliferative alterations triggered by UCP2 are distinct from uncoupling. In conclusion, although no changes in body and adipose weights were observed, GSPE, DHA-OR and the combination of both compounds ameliorated diet-induced obesity unbalances via the regulation of lipid metabolism implying AMPK regulation, observed in all of the treaded groups and suggesting a potential role for obesity-mediated metabolic diseases treatment.
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ACCEPTED MANUSCRIPT Acknowledgements This work was supported by grants AGL 2008–00387/ALI and AGL2011-23879
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from the Spanish Government.
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Conflict of interest
The authors declare that no conflict of interest exists.
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[48] Pecqueur C, Alves-Guerra C, Ricquier D, Bouillaud F. UCP2, a metabolic sensor coupling glucose oxidation to mitochondrial metabolism? IUBMB Life 2009;61:762–7.
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Figure legends
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Figure 1. Effect of proanthocyanidins and/or DHA supplementation on
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body weight and food intake in obese rats A. Increase of body weight during the treatment. B. Food intake during the entire treatment. Rats were fed cafeteria diet (CD) or standard diet (STD) for 10 weeks. After 10 weeks, rats were treated orally with vehicle (CD), 25 mg GSPE/kg body weight (CD+GSPE), 500 mg DHA/kg body weight (CD+DHAOR) or 25 mg GSPE/kg body weight +500 mg DHA/kg body weight (CD+GSPE+DHA-OR) for 21 days, concomitant with CD. Body weight and food intake was monitored regularly during the experiment. Data are means ± SEM of seven animals per group. * denote a significant difference between groups
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ACCEPTED MANUSCRIPT by ANOVA analysis (p< 0.05). The dashed line indicates the baseline values on
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the standard diet.
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Figure 2. Effect of proanthocyanidins and/or DHA supplementation on RQ
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values in obese rats
A. RQ values at 10 days of treatment. B. RQ values at 20 days of treatment.
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indirect calorimetry during 6-hours in postprandial state Experimental details as in figure 1.RQ, respiratory quotient. Data are means ± SEM of seven animals per group. ** denote a significant difference between all treated groups by ANOVA analysis (p< 0.01). * denote a significant difference between all treated
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groups by ANOVA analysis (p< 0.05). The dashed line indicates the baseline
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values on the standard diet.
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Figure 3. Effect of proanthocyanidins and/or DHA supplementation on
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plasma adipocytokines profiles in obese rats A. Adiponectin B. Leptin. Experimental details as in figure 1. Data are means ± SEM of seven animals per group. a and b denote a significant difference between groups by ANOVA analysis (p< 0.05). The dashed line indicates the baseline values on the standard diet.
Figure 4. Effect of proanthocyanidins and/or DHA supplementation on plasma glucose and insulin profiles in obese rats A.Glucose B. Insulin C. HOMA index. Experimental details as in Figure 1. Data are means ± SEM of seven animals per group. a and b denote a significant
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indicates the baseline values on the standard diet.
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Figure 5. Effect of proanthocyanidins and/or DHA supplementation on gene expression in the skeletal muscle of obese rats
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mRNA gene expression of A. Pparα. B. ATPase. C. Ucp3. D. Mnsod. E. Ucp2.
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F. Cpt1b. G. Lpl. H. Cd36. Experimental details as in figure 1. Data are means ± SEM of seven animals per group. a and b denote a significant difference between groups by ANOVA analysis (p< 0.05). The dashed line indicates the baseline values on the standard diet.
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Figure 6. Effect of proanthocyanidins and/or DHA supplementation on
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AMPK phosphorylation in the skeletal muscle of obese rats p-AMPK/AMPK protein levels. Experimental details as in figure 1. Data are
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means ± SEM of seven animals per group. a, b and c denote a significant
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difference between CD, CD+GSPE, CD+ DHA-OR group respectively by ANOVA analysis(p< 0.05). The dashed line indicates the baseline values on the standard diet.
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Fig. 1
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ACCEPTED MANUSCRIPT Table 1. Effect of proanthocyanidins and/or DHA supplementation on adipose
CD
CD+GSPE
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STD
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tissue weight
CD+DHA-OR
CD+GSPE+ DHA-OR
14.14 ± 1.34a 26.14 ± 2.24b 25.29 ± 1.31b
25.39 ± 2.53b
33.00 ± 4.31b
perirrenal (g tissue / g bw)
20.52 ± 0.70a 37.81 ± 3.18b 38.14 ± 1.94b
38.65 ± 2.76b
44.60 ± 3.40b
epididymal (g tissue / g bw)
24.80 ± 2.20a 40.16 ± 3.61b 35.47 ± 1.94ab
43.79 ± 5.61b
45.43 ± 3.40b
adiposity index
5.80 ± 0.27a
10.86 ± 0.99b
12.30 ± 1.09b
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mesenteric (g tissue / g bw)
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10.40 ± 0.82b 9.89 ± 0.23b
STD, Standard diet; CD, cafeteria diet; CD+GSPE, cafeteria diet + Grape seed proanthocyanidin extract; CD+DHA-OR, cafeteria diet+docosahexaenoic acid oil rich extract; CD+GSEP+DHA-OR, diet + Grape seed proanthocyanidin extract + docosahexaenoic acid oil
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rich extract. Data are means ± SEM of seven animals per group. a and b denote a significant
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difference (p< 0.05) between groups by ANOVA analysis.
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ACCEPTED MANUSCRIPT Table 2: Effect of proanthocyanidins and/or DHA supplementation on oxygen consumption by skeletal muscle mitochondria in obese rats CD+GSPE
CD+DHA-OR
CD+GSPE+DHA-OR
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CD
protocol pyruvate-malate
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Oxygen consumption flux (nmols O2 / mg*min)
State 2 (+Pyr)
48.7 ± 31.2a
55.3 ± 4.4a
State 3 (+ADP)
72.3 ± 35.9a
103.6 ± 43.7a
604.7 ± 100.0b
173.6 ± 9.3a
State 4 +oligomycin)
18.4 ± 7.6a
33.3 ± 7.4ab
73.2 ± 8.5b
44.5 ± 4.6ab
State U (+FCCP)
47.5 ± 22.8a
154.2 ± 60.0b
623.4 ± 75.3b
291.7 ± 40.3b
State 2 (+GM)
28.7 ±3.9a
34.0 ± 9.3ab
77.1 ± 0.9b
32.6 ± 6.4a
State 3 (+ADP)
33.3 ± 20.6a
107.7 ± 12.9a
182.1 ±25.0b
150.6 ± 37.3ab
State 3 (+rot)
17.4 ± 7.2ab
24.9 ± 3.5a
47.5 ± 6.5b
20.9 ± 5.6a
State 3 (+Succ)
64.9 ± 22.7a
102.7 ± 17.4ab
181.8 ± 18.4b
82.0 ± 16.0a
State 4 (+oligomycin)
56.9 ± 20.2a
74.0 ± 10.0a
136.2 ± 11.1b
48.4 ± 8.2a
63.5 ± 9.1a
167.4 ± 12.2b
54.5 ± 11.4a
8.7 ± 2.3a
42.4 ± 10.9ab
57.1 ± 9.1b
21.1 ± 8.4ab
State 2 (+CPCoA)
10.0 ± 6.5a
85.8 ± 27.9b
64.1 ± 10.2ab
27.6 ± 6.3a
State 3 (+ ADP)
9.0 ± 6.3a
150.2 ± 45.8ab
231.2 ± 29.8b
39.6 ± 12.5a
State 4 (+oligomycin)
2.7 ± 1.1a
76.9 ± 23.6b
97.8 ± 17.7ab
28.3 ± 6.3a
State U (+FCCP)
2.0 ± 1.1a
103.5 ± 53.5b
243.3 ± 60.7c
60.0 ± 33.3b
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State U ( +FCCP)
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protocol GMS(r)
43.6 ± 12.9a
67.4 ± 6.4a
40.9 ± 7.6a
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State 2 (+C)
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protocol Palmitoyl-CoA
STD, Standard diet; CD, cafeteria diet; CD+GSPE, cafeteria diet + Grape seed proanthocyanidin extract; CD+DHA-OR, cafeteria diet+docosahexaenoic acid oil rich extract; CD+GSEP+DHA-OR, diet + Grape seed proanthocyanidin extract + docosahexaenoic acid oil rich extract;Pyr, pyruvate; GM, Glutamate-Malate; rot, rotenone; succ, succiante; c, carnitine; CPCoA, carnitine + palmitoyl-CoA. Data are means ± SEM of seven animals per group. a,b and c denote significant differences between groups by ANOVA analysis (p< 0.05).
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ACCEPTED MANUSCRIPT Table 3: Effect of proanthocyanidins and/or DHA supplementation on the mitochondrial enzymatic activities and skeletal muscle damage of obese
CD+GSPE
Mitochondrial enzymatic activities (µmols/min*mg protein)
CD+DHA-OR
CD+GSPE +DHA-OR
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CD
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rats
12.6±4.9a
4.8 ± 0.5a
5.1±1.1ab
208.0±36.2ab
145.6±22.3b
19.5 ± 11.7a
10.1±3.3a
citrate synthase (CS)
65.7 ± 24.0a
122.9 ± 4.8ab
130.6 ± 17.1ab
181.9±40.9b
ATPase
5.6 ± 1.3ab
8.8± 2.7b
Skeletal muscle damage parameter 261.5±17.3a
159.4±27.1b
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CK (U/L)
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cytochrome c oxidase (COX) 18.7 ± 4.4a
STD, Standard diet; CD, cafeteria diet; CD+GSPE, cafeteria diet + Grape seed proanthocyanidin extract; CD+DHA-OR, cafeteria diet+docosahexaenoic acid oil rich extract; CD+GSEP+DHA-OR, diet + Grape seed proanthocyanidin extract + docosahexaenoic acid oil rich extrac; COX, cytochrome c oxidase; CS, citrate synthase; CK, creatine kinase. Data are
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means ± SEM of seven animals per group. a and b denote a significant difference (p< 0.05)
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S0955-2863(14)00113-2 doi: 10.1016/j.jnutbio.2014.05.003 JNB 7207
To appear in:
The Journal of Nutritional Biochemistry
Received date: Revised date: Accepted date:
13 January 2014 15 April 2014 2 May 2014
Please cite this article as: Casanova Ester, Baselga-Escudero Laura, Ribas-Latre Aleix, Arola-Arnal L´ıdia Ced´o Anna, Pinent Montserrat, Blad´e Cinta, Arola Llu´ıs, Josepa Salvad´o M, Chronic intake of proanthocyanidins and docosahexaenoic acid improves skeletal muscle oxidative capacity in diet-obese rats, The Journal of Nutritional Biochemistry (2014), doi: 10.1016/j.jnutbio.2014.05.003
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ACCEPTED MANUSCRIPT
Chronic intake of proanthocyanidins and docosahexaenoic acid
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improves skeletal muscle oxidative capacity in diet-obese rats
Ester Casanova, Laura Baselga-Escudero, Aleix Ribas-Latre, Lídia Cedó Anna Arola-Arnal, Montserrat Pinent, Cinta Bladé, Lluís Arola, M. Josepa
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Salvadó*
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Grup de Nutrigenomica. Departament de Bioquimica i Biotecnologia. Universitat Rovira i Virgili. Campus Sescel·lades, 43007. Tarragona. Spain Corresponding Author: M. Josepa Salvadó
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Department of Biochemistry and Biotechnology,
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Univeristat Rovira i Virgili
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C/ Marcel·lí i Domingo s/n 43007 Tarragona
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Spain
Phone: +34977559567 Fax: +34977558232 E-mail: [email protected]
Running title: proanthocyanidins and omega3 improve muscle oxidation:
Keywords: obesity, docosahexaenoic acid, proanthocyanidins, skeletal muscle, mitochondria, β-oxidation
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ACCEPTED MANUSCRIPT Abbreviations AMPK (5’-AMP-activated protein kinase), Cafeteria diet (CD), CK (creatine
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kinase), COX (Cytrochrome c oxidase) ,CPT ( palmitoyl translocase) ,CS
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(citrate synthase), DHA (docosahexaenoic acid), EPA ( eicosapentanoic acid), ETC (electron transport chain), FA (fatty acid), FCCP (trifluorocarbonylcyanide
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phenylhydrazone), GSPE (grape seed proanthocyanidins extract, Mnsod (manganes superoxide), OXPHOS (oxidative phosphorylation), PPARs
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(peroxisome proliferator-activated receptors), PUFAs (polyunsaturated fatty acids), RQ (respiratory quotient), STD (standard diet),TCA ( tricarboxylic acid
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cycle).
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ACCEPTED MANUSCRIPT Abstract Obesity has become a worldwide epidemic. The cafeteria diet (CD) induces
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obesity and oxidative stress-associated insulin resistance. Polyunsaturated fatty
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acids and polyphenols are dietary compounds that are intensively studied as products that can reduce the health complications related to obesity. We
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evaluate the effects of 21 days of supplementation with grape seed
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proanthocyanidins extract (GSPE), docosahexaenoic-rich oil (DHA-OR) or both compounds (GSPE+DHA-OR) on skeletal muscle metabolism in diet-obese rats. The supplementation with different treatments did not reduce body weight although all groups used more fat as fuel, particularly when both products were
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co-administered; muscle β-oxidation was activated, the mitochondrial functionality and oxidative capacity was higher, and fatty acid uptake gene
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expressions were upregulated. In addition to these outcomes shared by all treatments, GSPE reduced insulin resistance, and improved muscle status.
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Both treatments increased 5’-AMP-activated protein kinase (AMPK)
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phosphorylation, which was consistent with higher plasma adiponectin levels. Moreover AMPK activation by DHA-OR was also correlated with an upregulation of peroxisome proliferator-activated receptor alpha (Pparα). GSPE+DHA-OR, in addition to activate AMPK and enhance fatty acid oxidation, increased the muscle gene expression of uncoupling protein 2 (Ucp2). In conclusion GSPE+DHA-OR induced modifications that improved muscle status and could counterbalance the deleterious effects of obesity, and such modifications are mediated at least in part, through the AMPK signaling pathway.
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ACCEPTED MANUSCRIPT Introduction Obesity is a complex, multifactorial disease characterized by an increase in
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body weight or, more specifically, an increase in adipose tissue. It is prevalent
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in both developed and developing countries and affects children as well as adults[1]. Obesity produces adverse health consequences, such as
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dyslipidemia, insulin resistance, hypertension, type 2 diabetes and
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cardiovascular disease. To reduce food intake and increase physical activity are the main strategies followed to prevent and reduce obesity. New strategies for weight control management contemplate the use of bioactive compounds in the diet providing potential antiobesity effects. In this sense, medium chain
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triglycerides increase energy expenditure and fatty acid oxidation [2,3], omega3 fatty acids reduce adiposity index [4], and polyphenols reduce adipose index,
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inflammation, and stimulate thermogenesis [5–7]. Skeletal muscle has an
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important role in the overall utilization and oxidation of fatty acids (FAs). Specifically, skeletal muscle manifests reduced reliance upon FA oxidation in
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obesity compared to skeletal muscle of lean individuals. Biochemical marker levels are consistent with these findings, i.e. Carnitine palmitoyl translocase (CPT) 1 expression is diminished in obesity, with reduced capacity for FA oxidation[8]. Obesity may also interfere with mitochondrial bioenergetics by affecting cellular respiratory functions and oxidative pathways. In addition, there is a chronic elevation of circulating FA levels, a reduction in the expression of genes involved in mitochondrial biogenesis, an increased production of reactive oxygen species, and an impaired mitochondrial biogenesis and function in skeletal muscle [9,10]. The cafeteria diet (CD) is a robust model of human metabolic syndrome compared to traditional lard-based high-fat diets [6]. In this 4
ACCEPTED MANUSCRIPT model, animals are fed with a standard diet (STD) and concurrently offered highly palatable, energy-dense foods ad libitum. This diet promotes voluntary
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hyperphagia [11] that results in rapid weight gain and increases fat pad mass
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and prediabetic parameters, such as glucose and insulin intolerance and dyslipidemia [12].
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The use of dietary compounds that reduce health complications related to these
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pathologies is emerging as a new approach. Previous reports have raised the possibility that polyphenols and omega-3 polyunsaturated fatty acids (PUFAs) are good dietary compounds for reducing obesity-induced metabolic syndrome [13–16]. Treatment of rodents with different proanthocyanidins results in
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activation of -oxidation [17,18], regulation of expression/activity of enzymes of lipogenesis and fatty acid oxidation[19], and upregulation of genes related to
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oxidative phosphorylation [17]. Likewise, omega-3 PUFAs are involved in a
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variety of mitochondrial processes. Particularly, docosahexaenoic acid (DHA) consumption, stimulated genes related to mitochondrial functionality, FA
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oxidation and inhibited genes involved in FA synthesis [20]. Moreover, they are natural ligands of PPARs, such as PPARα that is activated by several PUFAs [21,22], with eicosapentaenoic acid (EPA) and DHA as the predominantly active biological components [23,24]. A synergistic effect between polyphenols and omega-3 PUFAs has been shown [25]. In the present study, we considered the possibility that intake of grape seed proanthocyanidins extract (GSPE) and/or DHA-rich oil (DHA-OR) concomitant with a CD could influence muscle fat and carbohydrate oxidation rates, while improving the deleterious effects associated with obesity-induced metabolic syndrome. Our hypothesis is that the consumption of GSPE and 5
ACCEPTED MANUSCRIPT DHA-OR could increase mitochondrial oxidative capacity in skeletal muscle on obesity. Besides, AMPK activation activates fatty acid oxidation, insulin
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signaling and could be modulated by adipokines such as leptin and adiponectin,
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important regulators of whole-body energy metabolism [26,27] . Given the key role of AMPK on energy homeostasis, we hypothetized that the effects of the
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bioactive compounds could be though affecting hormonal control and through
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modulating the enzyme AMPK.
Thus, we evaluated the effect of individual or simultaneous administration of GSPE and DHA-OR by analyzing the respiratory quotient (RQ) ratio (using indirect calorimetry (IC)), insulin resistance, plasma adipokine levels,
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mitochondrial functionality (measuring oxygen consumption of isolated mitochondria from skeletal muscle), mitochondrial enzymatic activities and
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expression of key metabolic genes .
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Materials and Methods Grape seed proanthocyanidins extract GSPE was provided by Les Dérives Résiniques et Terpéniques (Dax, France). The GSPE composition used in this study has been previously analyzed [28]: catechin (58 µmol/g), epicatechin (52 µmol/g), epigallocatechin (5.50 µmol/g), epicatechin gallate (89 µmol/g), epigallocatechin gallate (1.40 µmol/g), dimeric procyanidins (250 µmol/g), trimeric procyanidins (1568 µmol/g), tetrameric procyanidins (8.8 µmol/g), pentameric procyanidins (0.73 µmol/g) and hexameric procyanidins (0.38 µmol/g).
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ACCEPTED MANUSCRIPT Oil rich in docosahexaenoic acid The DHA-OR was provided by Market DHATm-S (Columbia, MD, USA). The
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nutritional composition of the oil derived from marine alga, Schizochytrium sp., a
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rich source of DHA with sunflower lecithin anad rosemary extract (flavoring). The fatty acid profile of DHA-OR was 5.6% of 14:0, 16.1% of 16:0, 0.9% of
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18:0, 15% of 18:1n-9, 1.4% of 18:2n-6, 0.5% of 20:4n-6 (ARA), 1.1% of 20:5n-3
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(EPA), 16.2% of 22:5n-6 (DPA), 38.8% of 22:6n-3 (DHA) and 0.5% of others. The oil also contained tocopherols and ascorbyl palmitate as antioxidants to provide stability.
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Animals and diets
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Male Wistar rats weighing 150 g were supplied by Charles River Laboratories (Barcelona, Spain). The animals were housed in a 12 h light-dark-cycle at 21-
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23ºC. Following adaptation week, animals were divided into five equal groups (7
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rats/group) and housed individually to control the food intake. One group was the STD group, fed with standard chow diet (Panlab 04, Barcelona, Spain) ad libitum,and the remaining 4 groups were fed with STD plus CD consisting of hypercaloric diet with industrially processed foods designed for human consumption (cookies, cheese portions, bacon, foie-gras, sugary milk, cupcakes and carrots) composed of 35.20% carbohydrate, 23.40% fat, 11.70% protein, and 28.39 % water and 1.31% fiber. Animals were allowed free access to standard chow and water in addition to CD ad libitum. During the day, food was removed. Food consumption and weight gain were monitored weekly for 10 weeks until animals were 20% overweight compared with the STD group. Subsequently, different treatments were administered jointly with the CD for 21 7
ACCEPTED MANUSCRIPT days where food consumption and body wegith were monitored every 5 days. One group (CD+GSPE) was supplemented with 25 mg GSPE /kg body weight
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dissolved in 5% Arabic gum (Sigma-Aldrich, Madrid, Spain). The second group
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was supplemented with 500 mg oil-rich DHA (38.8%) / kg body weight dissolved in 5% of Arabic gum (CD+DHA-OR). The third group received GSPE (25 mg/kg
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body weight) plus 500 mg oil-rich DHA (38.8%)/ kg body weight dissolved in 5% of Arabic gum (CD+GSPE+DHA-OR)). All groups received the same volume of
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Arabic gum. The rats were treated in the afternoon, and then allowed free access to fresh portions of chow and/or CD during the night. On day 21 of treatment, all rats were fasted for 3-hours before being anesthetized using 50 mg/kg body weight of sodium pentobarbital (Fagron Iberica, Terrasa, Spain)
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and sacrificed by abdominal aorta exsanguination. Blood was collected using
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heparin (Deltalab, Barcelona, Spain). Plasma was obtained by centrifugation (1500 x g, 15 min, 4ºC) and stored at -80ºC until the subsequent plasma
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parameter analysis. Gastronecmius muscles were excised, weighed,
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immediately frozen in liquid N2 and stored at -80ºC until further analysis. Small portions of the gastrocnemius skeletal muscle were also placed on ice and used to isolate mitochondria for enzymatic assays and mitochondrial functionality. Mesenteric, perirrenal, and epydimidal adipose tissues were excised and weighed. All procedures were approved by the Animal Ethics Committee of our university.
Plasmatic Parameters Creatine Kinase (CK) and Glucose were measured using enzymatic colorimetric kits (QCA, Barcelona, Spain). Insulin was assayed by ELISA method following 8
ACCEPTED MANUSCRIPT the manufacturer’s instructions (Mercodia, Uppsala, Sweden). The HOMA index was calculated using the fasting values of glucose and insulin with the following
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formula:
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Adiponectin and Leptin levels were quantified using specific EIAs according to
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the manufacturer’s instructions (Biosource International, Inc, USA).
Indirect calorimetry
Assessment of respiratory metabolism and dates of analysis were performed in
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a ventilated hood system (Panlab Harvard Apparatus, Barcelona, Spain). RQ
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values (VO2 / VCO2) were recorded by IC measurements at 10 and 20 days of
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treatment during 6-hours of the postprandial period (9.00 h – 15.00 h).
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Mitochondrial respiration by high-resolution tracking The piece of gastrocnemius muscle placed in ice-cold homogenization medium buffer (100 mM sucrose, 50 mM KCl, 20 mM K+-TES, 1 mM EDTA and 0.2% (w/w) FA free-BSA (Sigma-Aldrich, Madrid, Spain) was used for mitochondrial respiratory assays. Mitochondria used for respiratory assays were isolated by differential centrifugations as described by Hoeks, J. [29]. Mitochondrial functionality was recorded at 37ºC, under stirring at 350 rpm, by a polarographic oxygen sensor in a two-chamber Oxygraph (OROBOROS® Instruments, Innsbruck, Austria). The oxygen flux was expressed in nmol O 2 per mg mitochondrial protein per minute. 9
ACCEPTED MANUSCRIPT To view the bioenergetics in freshly prepared mitochondria, we modulated oxidative phosphorylation (OXPHOS) function in the following ways: basal state
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(State2), the OXPHOS coupling effect (State3) stimulated with +450 µM ADP
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(Sigma-Aldrich, Madrid, Spain), non-phosphorylating respiration (State4) with the inhibition of ATP synthesis by the addition of 1 µg/ml oligomycin, and
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OXPHOS non-coupling effect (StateU) with +0.5 µM carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich, Madrid, Spain)
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addition to obtain the maximal respiratory capacity. The measurement of the OXPHOS system and the operation of the tricarboxylic acid cycle (TCA) were carried out using 3 different sets of substrates: a) 5 mM pyruvate +2.5 mM malate (glycolytic substrates) to show the functionality of the pyruvate
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dehydrogenase and TCA functionality; b)10 mM glutamate (complex I
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substrate) +10 mM succinate (complex II substrate) amino acids substrates to activate mitochondria respiration were used in combination of 0.5 µM of
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rotenone to inhibit complex I in order to study complex II capacity alone; c) 2
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mM L-carnitine +50 µM palmitoyl-CoA (FA substrate to induce β-oxidation). Briefly, 0.2 mg of protein where used by pyruvate-malate and glutamate-malate -succinate sets; by the other hand 0.5 mg of protein were used in carnitine+palmitoyl-CoA set and each set incubated in 2 ml respiration buffer.
Mitochondrial enzymatic activity Pieces of gastrocnemius were weighed and placed on ice for subsequent mitochondrial fraction isolation to analyze cytochrome c oxidase (COX), citrate synthase (CS) and ATPase activities according to the methods of Fuster et
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ACCEPTED MANUSCRIPT al.[30], Srere [31] and Bergmeyer, respectively, as we have previously
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detailed[32].
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RNA extraction and quantitative real-time PCR (qRT-PCR) analysis Total RNA was isolated from the gastrocnemius skeletal muscle tissue as
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detailed previously by Casanova et al.[33]. Specific Taqman Assay-on-Demand
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Probes were used: Peroxisome proliferator activated receptor alpha (Pparα) (Rn00566193_m1), Carnitine palmitoyltransferase 1b (Cpt1b) (Rn005664242_m1), Uncoupling protein 3 (Ucp3) (Rn00565874_m1), Uncoupling protein 2 (Ucp2) (Rn00571166_m1), ATPsynthase (Atpase)
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(Rn01527025_m1), Manganese Superoxide dismutase (Mnsod) (Rn00566942_g1), Lipoprotein Lipase (Lpl) (Rn00561482_m1), FA translocator
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CD36 (Cd36) (Rn00580728_m1). Cyclophilin peptidylprolyl isomerase A (Ppia)
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was used as an endogenous control gene (Rn00690933_m1). The relative
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mRNA expression levels were calculated using the 2-∆∆Ct method.
Western Blot (WB) analyses Protein was extracted from the frozen gastrocnemius skeletal muscle in RIPA lysis buffer (150 mM Tris-HCl, 1% Triton x-100, SDS 0.1%, 167 mM NaCl, 0.5% Na-deoxycholate) with a protease inhibitor cocktail 1/1000 (Sigma –Aldrich St. Louis, MO, USA), PMSF 1/100 (FLUKA-Biochemika, Switzerland) and a phosphatase inhibitor cocktail: cocktail II 1/100, cocktail III 1/100 (SigmaAldrich St.Louis, MO, USA). Total protein levels were determined using the Bradford method[34]. Proteins were loaded and run on a 10% SDSpolyacrylamide gel. Samples were transferred to PVDF membrane (Bio-Rad, 11
ACCEPTED MANUSCRIPT Hercules, CA, USA) using a transblot apparatus (Bio-Rad, Hercules, CA, USA) and subsequently blocked at room temperature with 5% (wt/vol) non-fat milk in
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TBS-T buffer (Tris-Buffered saline, 0.5% Tween-20). Primary antibodies were
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incubated overnight with shaking at 4ºC: rabbit AMPKα primary antibody, phospho AMPK α primary antibody (Cell Signaling Technology, Beverly, MA,
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USA), or rabbit α-tubulin (Sigma- Aldrich St. Louis, MO, USA). Blots were washed in TBST buffer and incubated with peroxidase-conjugated anti-rabbit
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secondary antibody (GE Healthcare, Buckinghamshire, UK) 1-hour. . Immunoreactive proteins were visualized using chemiluminiscence substrate kit ECL PLUS (GE Healthcare Buckinghamshire, UK) according to manufacturer’s instructions. Digital images were taken with a GBOX Chemi XL 1.4 image
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system (Syngene Ozyme, France). Quantification content was carried out in
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Image J Software (NIH, USA). The results were expressed as the relative
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intensity of p-AMPK/AMPK.
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Statistical Analysis
The results are expressed as the mean ± SEM of seven animals. SPSS Statistics version 19 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Significant differences were analyzed by one-way ANOVA followed by the Tukey post-hoc test. A p-value ≤ 0.05 was considered statistically significant.
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ACCEPTED MANUSCRIPT Results Food intake changed significantly only at specific points along the 21
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days of treatment
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CD significantly increased body weight as compared to STD at 2 weeks. The difference was more pronounced at 10 weeks (STD: 439.14±10.35a; CD:
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518.50±6.97b). Moreover, RQ values were significantly lower in CD than in STD
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group (STD: 1.00±0.01a; CD: 0.88±0.01b), which indicated a preference for lipid oxidation. Table 1 shows that mesenteric, perirenal and epididymal adipose tissue weights were higher in CD groups, than in STD group. Figure 1A shows body weight measurements during the treatment with no
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differences between groups. Figure 1B shows the food intake of each group during the entire treatment. At day 5 of treatment, the CD+GSPE+DHA-OR
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group increased their food intake significantly compared to the control CD
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group. Conversely, after 21 days, food intake was significantly decreased in the
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CD+GSPE group compared to CD group.
GSPE+DHA-OR administration along with CD decreased RQ values At 10 days of treatment (Figure 2A), RQ values from the CD group were significantly higher throughout the postprandial period compared to the treated groups. In contrast, the CD+GSPE+DHA-OR group had significantly lower RQ values and were also lower than those of the STD group. Figure 2B shows that the differences in RQ values between the CD groups are reduced at 20 days of treatment. The RQ was significantly lower in the CD+GSPE+DHA-OR group
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ACCEPTED MANUSCRIPT than in the remaining groups only at the end of the measurements (postprandial
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5-6-hours).
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GSPE and DHA-OR administration increased adiponectin levels
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Plasma adiponectin levels were significantly higher in CD+GSPE and CD+DHAOR groups compared to the CD group, while no significant differences were
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found in other treatment groups (Figure 3A). Moreover, leptin levels (Figure 3B) were significantly increased after CD+GSPE+DHA-OR administration compared
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to the other treatments.
GSPE administration reversed insulin resistance induced by CD
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Glucose plasma values did not differ between the CD and treated groups
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although all of them were increased compared to the values for the STD group (Figure 4A). Plasma insulin was decreased significantly in the CD+GSPE group,
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approaching the STD group values (Figure4B). The HOMA index (Figure 4C) was significantly decreased in the CD+GSPE group compared to the CD+DHAOR group, and was similar to STD group values.
GSPE and DHA-OR administration along with CD improved mitochondrial respiration compared to the CD group. The results of assessing the functionality of the OXPHOS system are described in Table 2. Animals fed with CD+DHA-OR had higher rates of respiration in all states compared to the other groups using three different sets of substrates in
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ACCEPTED MANUSCRIPT an aim to show the electron transport chain functionality between different sources of nutrients, especially in State3 which increased ATP production and
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in StateU that represents the respiratory maximum capacity. Mitochondrial
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respiration was lower in Palmitoyl-CoA oxidation set than in the other two substrate sets in the CD group. Moreover mitochondrial respiration in Palmitoyl-
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an increase of StateU by all treated groups.
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CoA oxidation set was increased in CD+GSPE and CD+DHA-OR groups with
Both GSPE and DHA-OR administration affected the expression of genes related to FA oxidation in skeletal muscle.
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Figure 5 shows that CD+DHA-OR treatment significantly increased Pparα, Cd36, Lpl and Cpt1b expression in muscle compared to the CD group.
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Additionally, CD+GSPE treatment significantly increased Cd36, Lpl and Cpt1b
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gene expression. For the CD+GSPE+DHA-OR group, Ucp2, Lpl and Cpt1b
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were significantly overexpressed. It is important to note that Ucp3 mRNA levels in rats fed a CD were higher than in those on the STD diet, although there were no differences between treated groups. None of the treatments altered ATPase or Mnsod gene expression.
GSPE administration increased ATPase activity, while GSPE and DHA-OR coadministration increased CS activity. COX activity showed no difference between groups. However, ATPase activity was higher in the CD+GSPE group compared to the other groups. CS activity in
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ACCEPTED MANUSCRIPT the CD+GSPE+DHA group was significantly elevated compared to the CD
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GSPE administration decreased CK plasma values
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group (Table3).
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CK, a plasma indicator of muscle damage, was significantly decreased by CD+GSPE administration with respect to the CD group, and also when
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administered concomitantly with DHA-OR. CK levels in both groups appeared to be restored to levels comparable to STD group (Table 3).
in skeletal muscle.
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AMPK phosphorylation was activated by GSPE or/and DHA-OR treatments
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Figure 6 shows that the administration of either GSPE or DHA-OR activated
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AMPK phosphorylation and significantly increased the ratio of
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phosphorylated/unphosphorylated AMPK compared to CD.
Discussion
Due to its high palatability, CD allows rodents to become hyperphagic by reducing their control over their food intake [11]. As a result of 10 weeks of eating the hypercaloric CD ad libitum, Wistar rats become obese, showing a 20% increase in body weight with higher mesenteric, perirenal and epididymal adipose tissue weights; with an adiposity index approximately twice STD-fed rat. Furthermore, CD rats are hypertriglyceridemic, hypercholesterolemic, hyperinsulinemic, and hyperleptinemic, with a higher HOMA index than STD
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ACCEPTED MANUSCRIPT rats[35–37]. After 10 weeks of consuming CD, GSPE, DHA-OR or a combination of both were added to the CD for 21 days. None of these
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treatments significantly improved body weight nor leptinemia, but increased
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plasma adiponectin. It is well known that obesity is associated to high concentrations of plasma leptin [38], and in the current study, GSPE did not
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suppress the elevated levels of leptin induced by obesity as found in another study where polyphenols suppressed the increases in leptin levels in high-fat-
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fed mice[26]. The addition of GSPE to the CD lowered insulinemia and HOMA index, thus improving the insulin resistance shown by CD rats in addition to their decreased TGs plasma levels observed in previous studies [33,35,39,40]. Published data showed that phenolic compounds reduced hyperinsulinemia by
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increasing adiponectin secretion and expression in obese diabetic mice [41,42].
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Here, we observed that adiponectin, not only reduced hyperinsulinemia but also stimulated β -oxidation in skeletal muscle via AMPK . The increase in FA
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oxidation following CD+GSPE treatment also modulate mitochondrial function
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substantiated by the high-resolution respirometry outcomes. Higher oxygen consumption in State2 and State4 of the palmitoyl-CoA oxidation demonstrated the increase of FA oxidation rates in ex vivo isolated mitochondria . Moreover, there was a high facility to increase, StateU, the maximum respiratory capacity and adapt OXPHOS system in extreme conditions by pyruvate/malate and palmitoyl-CoA pathways in CD+GSPE treated rats. Likewise, Pajuelo et al. demonstrated that GSPE modulates mitochondrial function, by adapting the OXPHOS system and increasing FA oxidation, also in brown adipose tissue of CD-fed rats [32].
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ACCEPTED MANUSCRIPT In the case of CD+GSPE rats, the significantly elevated transcript levels of Lpl, Cd36, Cpt1b and higher ATPase activity in skeletal muscle, concomitant with
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the reduced TG levels in plasma, was in agreement with the role of GSPE in
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redirecting circulating TGs to skeletal muscle for fat oxidation and ATP synthesis [33].In addition, in a study with a similar obesity model in rats, GSPE
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improved both lipidemia and the fat content in the liver [15]. All of these changes are consistent with high circulating adiponectin levels in the CD+GSPE
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group, because it has been reported that adiponectin increases the expression of molecules involved in FA transport (CD36) and combustion (acyl-coenzyme A oxidase) in skeletal muscle[38]. All these effects of GSPE underlie the
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prevention of muscle damage as shown by the lower plasma CK values. Likewise, DHA-OR increased plasma adiponectin levels, activated AMPK
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phopshorylation and elevated transcript levels of Lpl, Cd36, Cpt1b. In addition
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to increase β-oxidation in skeletal muscle, DHA-OR also increased mitochondrial OXPHOS functionality, with a maximum respiratory capacity
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shown in isolated mitochondria. In a previous study in which healthy rats were given an acute dose of DHA-OR plus a high fat overload, the results showed that DHA-OR facilitates the entry and uptake of long-chain FAs in skeletal muscle via Lpl and Cd36 overexpression, and increases the flux of FA oxidation to mitochondria by Cpt1b overexpression [33], as has been found in the present study with CD rats. This is likely to occur through upregulation of Pparα , because omega-3 PUFA are ligands of PPARα, resulting in a rapid upregulation of genes involved in lipid oxidation [43,44]; but may also be due to AMPK activation by increased adiponectin levels as other authors have reported [45–47]. 18
ACCEPTED MANUSCRIPT The effects of CD+GSPE+DHA-OR were similar to those of each separate compound in terms of muscle energy homeostasis and oxidative capacity. The
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increased mitochondrial oxidative capacity and fatty acid oxidation by GSPE
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and/or DHA-OR was reinforced by the results of RQ values at 10 days of treatment using a markedly higher percentage of fatty acid combustion over
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carbohydrates. Meanwhile, at the end of the treatment CD+GSPE+DHA-OR treated rats fuelled essentially FAs, in spite of a RQ ratio closer to the other
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groups, so the combination of both compounds caused the highest burning of fat overload. Again, GSPE+DHA-OR significantly upregulated gene expression of key markers of β-oxidation, Lpl and Cpt1b.These results agreed with the high functionality of mitochondria, and with the improvement of the unusual low
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oxygen consumption shown in CD group with palmitate as substrate. Once
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again, these effects occur through AMPK activation, which was concomitant with significantly elevated transcript levels of Ucp2. Pecqueur et al.[48] showed
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that UCP2 promotes mitochondrial FA oxidation while limiting mitochondrial
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catabolism of pyruvate, suggesting that the metabolic and proliferative alterations triggered by UCP2 are distinct from uncoupling. In conclusion, although no changes in body and adipose weights were observed, GSPE, DHA-OR and the combination of both compounds ameliorated diet-induced obesity unbalances via the regulation of lipid metabolism implying AMPK regulation, observed in all of the treaded groups and suggesting a potential role for obesity-mediated metabolic diseases treatment.
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ACCEPTED MANUSCRIPT Acknowledgements This work was supported by grants AGL 2008–00387/ALI and AGL2011-23879
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from the Spanish Government.
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Conflict of interest
The authors declare that no conflict of interest exists.
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Figure legends
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Figure 1. Effect of proanthocyanidins and/or DHA supplementation on
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body weight and food intake in obese rats A. Increase of body weight during the treatment. B. Food intake during the entire treatment. Rats were fed cafeteria diet (CD) or standard diet (STD) for 10 weeks. After 10 weeks, rats were treated orally with vehicle (CD), 25 mg GSPE/kg body weight (CD+GSPE), 500 mg DHA/kg body weight (CD+DHAOR) or 25 mg GSPE/kg body weight +500 mg DHA/kg body weight (CD+GSPE+DHA-OR) for 21 days, concomitant with CD. Body weight and food intake was monitored regularly during the experiment. Data are means ± SEM of seven animals per group. * denote a significant difference between groups
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ACCEPTED MANUSCRIPT by ANOVA analysis (p< 0.05). The dashed line indicates the baseline values on
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the standard diet.
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Figure 2. Effect of proanthocyanidins and/or DHA supplementation on RQ
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values in obese rats
A. RQ values at 10 days of treatment. B. RQ values at 20 days of treatment.
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indirect calorimetry during 6-hours in postprandial state Experimental details as in figure 1.RQ, respiratory quotient. Data are means ± SEM of seven animals per group. ** denote a significant difference between all treated groups by ANOVA analysis (p< 0.01). * denote a significant difference between all treated
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groups by ANOVA analysis (p< 0.05). The dashed line indicates the baseline
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values on the standard diet.
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Figure 3. Effect of proanthocyanidins and/or DHA supplementation on
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plasma adipocytokines profiles in obese rats A. Adiponectin B. Leptin. Experimental details as in figure 1. Data are means ± SEM of seven animals per group. a and b denote a significant difference between groups by ANOVA analysis (p< 0.05). The dashed line indicates the baseline values on the standard diet.
Figure 4. Effect of proanthocyanidins and/or DHA supplementation on plasma glucose and insulin profiles in obese rats A.Glucose B. Insulin C. HOMA index. Experimental details as in Figure 1. Data are means ± SEM of seven animals per group. a and b denote a significant
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Figure 5. Effect of proanthocyanidins and/or DHA supplementation on gene expression in the skeletal muscle of obese rats
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mRNA gene expression of A. Pparα. B. ATPase. C. Ucp3. D. Mnsod. E. Ucp2.
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F. Cpt1b. G. Lpl. H. Cd36. Experimental details as in figure 1. Data are means ± SEM of seven animals per group. a and b denote a significant difference between groups by ANOVA analysis (p< 0.05). The dashed line indicates the baseline values on the standard diet.
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Figure 6. Effect of proanthocyanidins and/or DHA supplementation on
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AMPK phosphorylation in the skeletal muscle of obese rats p-AMPK/AMPK protein levels. Experimental details as in figure 1. Data are
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means ± SEM of seven animals per group. a, b and c denote a significant
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difference between CD, CD+GSPE, CD+ DHA-OR group respectively by ANOVA analysis(p< 0.05). The dashed line indicates the baseline values on the standard diet.
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Fig. 1
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ACCEPTED MANUSCRIPT Table 1. Effect of proanthocyanidins and/or DHA supplementation on adipose
CD
CD+GSPE
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STD
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tissue weight
CD+DHA-OR
CD+GSPE+ DHA-OR
14.14 ± 1.34a 26.14 ± 2.24b 25.29 ± 1.31b
25.39 ± 2.53b
33.00 ± 4.31b
perirrenal (g tissue / g bw)
20.52 ± 0.70a 37.81 ± 3.18b 38.14 ± 1.94b
38.65 ± 2.76b
44.60 ± 3.40b
epididymal (g tissue / g bw)
24.80 ± 2.20a 40.16 ± 3.61b 35.47 ± 1.94ab
43.79 ± 5.61b
45.43 ± 3.40b
adiposity index
5.80 ± 0.27a
10.86 ± 0.99b
12.30 ± 1.09b
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mesenteric (g tissue / g bw)
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10.40 ± 0.82b 9.89 ± 0.23b
STD, Standard diet; CD, cafeteria diet; CD+GSPE, cafeteria diet + Grape seed proanthocyanidin extract; CD+DHA-OR, cafeteria diet+docosahexaenoic acid oil rich extract; CD+GSEP+DHA-OR, diet + Grape seed proanthocyanidin extract + docosahexaenoic acid oil
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rich extract. Data are means ± SEM of seven animals per group. a and b denote a significant
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difference (p< 0.05) between groups by ANOVA analysis.
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ACCEPTED MANUSCRIPT Table 2: Effect of proanthocyanidins and/or DHA supplementation on oxygen consumption by skeletal muscle mitochondria in obese rats CD+GSPE
CD+DHA-OR
CD+GSPE+DHA-OR
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CD
protocol pyruvate-malate
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Oxygen consumption flux (nmols O2 / mg*min)
State 2 (+Pyr)
48.7 ± 31.2a
55.3 ± 4.4a
State 3 (+ADP)
72.3 ± 35.9a
103.6 ± 43.7a
604.7 ± 100.0b
173.6 ± 9.3a
State 4 +oligomycin)
18.4 ± 7.6a
33.3 ± 7.4ab
73.2 ± 8.5b
44.5 ± 4.6ab
State U (+FCCP)
47.5 ± 22.8a
154.2 ± 60.0b
623.4 ± 75.3b
291.7 ± 40.3b
State 2 (+GM)
28.7 ±3.9a
34.0 ± 9.3ab
77.1 ± 0.9b
32.6 ± 6.4a
State 3 (+ADP)
33.3 ± 20.6a
107.7 ± 12.9a
182.1 ±25.0b
150.6 ± 37.3ab
State 3 (+rot)
17.4 ± 7.2ab
24.9 ± 3.5a
47.5 ± 6.5b
20.9 ± 5.6a
State 3 (+Succ)
64.9 ± 22.7a
102.7 ± 17.4ab
181.8 ± 18.4b
82.0 ± 16.0a
State 4 (+oligomycin)
56.9 ± 20.2a
74.0 ± 10.0a
136.2 ± 11.1b
48.4 ± 8.2a
63.5 ± 9.1a
167.4 ± 12.2b
54.5 ± 11.4a
8.7 ± 2.3a
42.4 ± 10.9ab
57.1 ± 9.1b
21.1 ± 8.4ab
State 2 (+CPCoA)
10.0 ± 6.5a
85.8 ± 27.9b
64.1 ± 10.2ab
27.6 ± 6.3a
State 3 (+ ADP)
9.0 ± 6.3a
150.2 ± 45.8ab
231.2 ± 29.8b
39.6 ± 12.5a
State 4 (+oligomycin)
2.7 ± 1.1a
76.9 ± 23.6b
97.8 ± 17.7ab
28.3 ± 6.3a
State U (+FCCP)
2.0 ± 1.1a
103.5 ± 53.5b
243.3 ± 60.7c
60.0 ± 33.3b
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protocol GMS(r)
43.6 ± 12.9a
67.4 ± 6.4a
40.9 ± 7.6a
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State 2 (+C)
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protocol Palmitoyl-CoA
STD, Standard diet; CD, cafeteria diet; CD+GSPE, cafeteria diet + Grape seed proanthocyanidin extract; CD+DHA-OR, cafeteria diet+docosahexaenoic acid oil rich extract; CD+GSEP+DHA-OR, diet + Grape seed proanthocyanidin extract + docosahexaenoic acid oil rich extract;Pyr, pyruvate; GM, Glutamate-Malate; rot, rotenone; succ, succiante; c, carnitine; CPCoA, carnitine + palmitoyl-CoA. Data are means ± SEM of seven animals per group. a,b and c denote significant differences between groups by ANOVA analysis (p< 0.05).
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ACCEPTED MANUSCRIPT Table 3: Effect of proanthocyanidins and/or DHA supplementation on the mitochondrial enzymatic activities and skeletal muscle damage of obese
CD+GSPE
Mitochondrial enzymatic activities (µmols/min*mg protein)
CD+DHA-OR
CD+GSPE +DHA-OR
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CD
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12.6±4.9a
4.8 ± 0.5a
5.1±1.1ab
208.0±36.2ab
145.6±22.3b
19.5 ± 11.7a
10.1±3.3a
citrate synthase (CS)
65.7 ± 24.0a
122.9 ± 4.8ab
130.6 ± 17.1ab
181.9±40.9b
ATPase
5.6 ± 1.3ab
8.8± 2.7b
Skeletal muscle damage parameter 261.5±17.3a
159.4±27.1b
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CK (U/L)
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cytochrome c oxidase (COX) 18.7 ± 4.4a
STD, Standard diet; CD, cafeteria diet; CD+GSPE, cafeteria diet + Grape seed proanthocyanidin extract; CD+DHA-OR, cafeteria diet+docosahexaenoic acid oil rich extract; CD+GSEP+DHA-OR, diet + Grape seed proanthocyanidin extract + docosahexaenoic acid oil rich extrac; COX, cytochrome c oxidase; CS, citrate synthase; CK, creatine kinase. Data are
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means ± SEM of seven animals per group. a and b denote a significant difference (p< 0.05)
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