Food and Chemical Toxicology 49 (2011) 14261430

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Food and Chemical Toxicology

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Pomegranate seed oil, a rich source of punicic acid, prevents diet-induced obesity and insulin resistance in mice
Irene O.C.M. Vroegrijk a, Janna A. van Diepen a, Sjoerd van den Berg b, Irene Westbroek c, Hiskias Keizer d, Luisa Gambelli d, Raquel Hontecillas e, Josep Bassaganya-Riera e, Gerben C.M. Zondag c, Johannes A. Romijn a, Louis M. Havekes a, Peter J. Voshol a,f,
a

Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, Netherlands Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands DNage B.V., Leiden, Netherlands d Lipid Nutrition B.V., Wormerveer, Netherlands e Nutritional Immunology & Molecular Medicine Lab, Blacksburg, VA, USA f University of Cambridge, Metabolic Research Laboratories, Cambridge, UK
b c

a r t i c l e

i n f o

a b s t r a c t
Background: Pomegranate seed oil has been shown to protect against diet induced obesity and insulin resistance. Objective: To characterize the metabolic effects of punicic acid on high fat diet induced obesity and insulin resistance. Design: High-fat diet or high-fat diet with 1% Pomegranate seed oil (PSO) was fed for 12 weeks to induce obesity and insulin resistance. We assessed body weight and composition (pSABRE DEXA-scan), energy expenditure (Columbus Instruments) and insulin sensitivity at the end of the 12 weeks. Results: PSO intake resulted in a lower body weight, 30.5 2.9 vs 33.8 3.2 g PSO vs HFD respectively, p = 0.02, without affecting food intake or energy expenditure. The lower body weight was fully explained by a decreased body fat mass, 3.3 2.3 vs 6.7 2.7 g for PSO and HFD fed mice, respectively, p = 0.02. Insulin clamps showed that PSO did not affect liver insulin sensitivity but clearly improved peripheral insulin sensitivity, 164 52% vs 92 24% for PSO and HFD fed mice respectively, p = 0.01. Conclusions: We conclude that dietary PSO ameliorates high-fat diet induced obesity and insulin resistance in mice, independent of changes in food intake or energy expenditure. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 23 November 2010 Accepted 20 March 2011 Available online 31 March 2011 Keywords: Punicic acid Insulin resistance Obesity Energy expenditure Metabolic exibility

1. Introduction Conjugated fatty acids such as conjugated linoleic acid (CLA) and conjugated linolenic acid (CLnA) have recently attracted significant attention because of their health benets in a variety of models of metabolic and chronic inammatory diseases. Punicic acid (PUA), also known as trichosanic acid, is a conjugated triene fatty acid naturally found at high concentrations in the seed of Punica granatum (Punicaceae, Pomegranate) (Sassano et al., 2009) and Trichosanthes kirilowii (Joh et al., 1995). PUA constitutes 6483% of the Pomegranate seed oil (PSO) (Ahlers et al., 1954; Kaufman and Wiesman, 2007). In addition to PUA, PSO also contains minor amounts of other CLnA isomers including a-eleostearic acid (ESA) and catalpic acid (CAT) (Sassano et al., 2009). Phytosterols (i.e.,
Corresponding author at: University of Cambridge, Metabolic Research Laboratories, Level 4, Institute of Metabolic Science, Box 289, Addenbrookes Hospital, Cambridge CB2 0QQ, UK. Tel.: +44 1223 769093; fax: +44 1223 330598. E-mail address: pv256@medschl.cam.ac.uk (P.J. Voshol).
0278-6915/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.03.037

beta-sitosterol, campesterol, and stigmasterol) are also found in quite a high concentration in the PSO (40896205 mg/kg of PSO) (Kaufman and Wiesman, 2007), which may contribute to the overall spectrum of health benets observed. Structurally, PUA is a conjugated octadecatrienoic acid, containing cis-9, trans-11, and cis-13 double bonds, that resembles the naturally occurring cis-9, trans-11 CLA isomer, the predominant CLA isomer in beef and milk (Bassaganya-Riera et al., 2002; OShea et al., 2004). CLA exerts its insulin-sensitizing and anti-inammatory actions through a mechanism dependent activation of peroxisome proliferator activated receptor a and c (PPAR c) (Moya-Camarena et al., 1999; Bassaganya-Riera et al., 2004). A recent report demonstrates that PSO consumption decreases weight gain and type 2 diabetes risk in CD-1 mice with diet2induced obesity (McFarlin et al., 2009). In line with these ndings, we have shown that PSO ameliorates glucose tolerance and suppresses obesity-related inammation (Hontecillas et al., 2009). In this study, by using a loss-of-function approach (i.e., immune cell specic PPAR c null mice), we demonstrated that some of the

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benecial effects of PSO are mediated by activation of PPAR c (Hontecillas et al., 2009). Others have found that PSO inhibits TNF-a-induced neutrophil hyperactivation and protects from experimental colonic inammation in rats by targeting the p38MAPKinase/Ser345-p47phox axis (Boussetta et al., 2009). PSO ameliorates inammation induced colorectal cancer in rats (Kohno et al., 2004a) and the ESA-rich bitter gourd seed oil ameliorates colonic carcinogenesis and upregulates colonic PPAR c expression in rats (Kohno et al., 2004b). Catalpic acid decreases abdominal fat deposition, improves glucose tolerance and upregulates adipose PPAR a in two mouse models of obesity (Hontecillas et al., 2008). Even though several studies have investigated the effect of PSO on glucose tolerance and other indicators of type 2 diabetes, none of the previous studies have utilized the hyperinsulinemic euglycemic clamp technology, which is considered the gold standard for assessing insulin resistance. By using this technology we demonstrate for the rst time that dietary PSO-supplementation does not affect liver insulin sensitivity but clearly improves peripheral insulin sensitivity under high-fat fed conditions. Furthermore, we show novel evidence that PSO increases the carbohydrate oxidative capacity. In conclusion, dietary PSO supplementation ameliorates diet-induced obesity and insulin resistance.

2.3. Indirect calorimetry After 10 weeks of HFD feeding groups of 8 mice per diet were subjected to individual indirect calorimetry measurements for a period of 4 consecutive days (Comprehensive Laboratory Animal Monitoring System, Columbus Instruments, Columbus Ohio, US) (van den Berg et al., 2010a, 2010b). A period of 24 h prior to the start of the experiment allowed for acclimatization of the animals to the cages and the single housing. Experimental analysis started at 09:00 h and continued for 72 h. Analyzed parameters included real time food and water intake, as well as meal size, frequency and duration. Oxygen consumption (VO2) and carbon dioxide production rates (VCO2) were measured at intervals of 7 min. Respiratory exchange ratio (RER) as a measure for metabolic substrate choice was calculated using the following formula:

RER VCO2 =VO2 :

Carbohydrate and fat oxidation rates were calculated from VO2 and VCO2 using the following formulas (Pronnet and Massicotte, 1991):

Carbohydrate oxidation kcal=h 4:585 VCO2 3:226 VO2 4=1000 Fat oxidation kcal=h 1:695 VO2 1:701 VCO2 9=1000
VO2 and VCO2 values are in mL/h. Total energy expenditure was calculated from the sum of carbohydrate and fat oxidation. Activity was monitored as infrared beam breaks in both X and Y axis. 2.4. Hyperinsulinemic euglycemic clamp experiments After 12 weeks on the high-fat diet, clamp experiments were performed as described previously (Goudriaan et al., 2005; Duivenvoorden et al., 2005; Voshol et al., 2001, 2003) after an overnight fast. Animals were anesthetized by intraperitoneal (ip) injection of a combination of 6.25 mg/kg acetylpromazine (Sano Sant Nutrition Animale, Libourne Cedex, France) 6.25 mg/kg midazolam (Roche, Mijdrecht, The Netherlands), and 0.31 mg/kg fentanyl (Janssen-Cilag, Tilburg, The Netherlands). An infusion needle was placed into the tail vein. After 45 min infusion of 3 D-[3- H]glucose at a rate of 0.8 lCi/h (specic activity, 620 GBq/mmol; Amersham, Little Chalfont, UK) to achieve steady-state levels, basal parameters were determined with 15-min intervals. Thereafter, a bolus of insulin (4.5 mU, Actrapid; Novo Nordisk, Chartres, France) was administered and the hyperinsulinemic clamp was started. Insulin was infused at a constant rate of 6.8 mU/h, and D-[3-3H]glucose was infused at a rate of 0.8 lCi/h. A variable infusion of 12.5% D-glucose (in PBS) was started to maintain blood glucose at approximately 5.0 mM. Blood glucose was measured with a AccuCheck Aviva hand glucose measurer (Disetronic Medical Systems, Vianen, The Netherlands) every 10 min to monitor glucose levels and accordingly adjust the glucose infusion pump. After reaching steady state, blood samples were taken at 10-min intervals during 30 min to determine steady-state levels of [3H]glucose. After the last blood sample, mice were killed by cervical dislocation and the organs were dissected and immediately frozen. An average clamp experiment took approximately 3 h, and anesthesia was maintained throughout the procedure. Hematocrit was not different before and after the clamp procedure. 2.5. Analysis of clamp samples Plasma insulin concentrations were measured by ELISA (ALPCO Diagnostics, Windham, NH). To measure plasma [3H]glucose, trichloroacetic acid (nal concentration 2%) was added to 7.5 ll plasma to precipitate proteins using centrifugation. The supernatant was dried to remove water and re-suspended in water. The samples were counted using scintillation counting (Packard Instruments, Dowers Grove, IL). 2.6. Calculations

2. Materials and methods 2.1. Animals and diets Male C57Bl/J6 mice were housed in a temperature controlled room on a 12-h light/dark cycle (lights on from 7:00 AM till 7:00 PM) and had free access to water and standard mouse chow diet. At the age of 10 weeks, they were put on a high fat diet, in which 45.3% of the energy is derived from fat (saturated palm oil) for a period of 12 weeks. Mice were randomly assigned to either one of two dietary intervention groups. The rst group (HFD) was maintained on the high-fat diet. The second group (PSO) was fed the same high fat diet as the control group, but per 100 g of food, 1 g of fat was replaced by Pomegranate seed oil, (Lipid Nutrition, Wormerveer, NL). Both diets were manufactured by Research Diet Services, Woerden, the Netherlands. See Table 1 for diet compositions. The study was approved by the institutions animal welfare committee, following Dutch guidelines for the use of laboratory animals.

2.2. Blood analysis Blood samples were taken before and after the hyperinsulinemic euglycemic clamp analysis, after an overnight (16 h) fast. Blood samples were taken from the mice by tail bleeding and collected in paraoxinized glass tubes (to prevent hydrolysis of triacylglycerols) (Bijland et al., 2010) and kept on ice. Then, the samples were spun (13,000 rpm) at 4 C for 4 min and the separated plasma was immediately assayed for glucose and free fatty acids (FFA). The remaining plasma was frozen in liquid nitrogen and stored at 20 C for later measurement of insulin. Plasma glucose was determined by a commercially available kit (Sigma diagnostics Inc., St. Louis, MO, USA). FFA was measured enzymatically with a NEFA-C kit (Wako Chemicals, GmbH, Neuss, Germany). Insulin concentrations were measured by using a radio immunoassay kit (ALPCO Diagnostics, Windham, NH).

Table 1 Composition of the diets (purchased from Research Diets, Woerden, The Netherlands). Variable Energy content (kJ/g) Macronutrients As% of energy Fat palm oil PSO Carbohydrates Proteins As% of weight Fat palm oil PSO Carbohydrates Proteins High fat diet 20.0 PSO diet 20.0

The glucose turnover rate (lmol/min kg) was calculated during the basal period and under steady-state clamp conditions as the rate of tracer infusion (dpm/min) divided by the plasma specic activity of [3H]glucose (dpm/lmol). The ratio was corrected for body weight. The hyperinsulinemic hepatic glucose production (HGP) was calculated as the difference between the tracer-derived rate of glucose appearance and the glucose infusion rate. 2.7. Body anthropometry and dual energy X-ray absorptiometry (DEXA) scan analysis Animals were subjected to DEXA scan analysis after energy expenditure assessment (week 10 of HFD feeding) in fed conditions to avoid weight loss induced by overnight fasting. Animals were weighed and sedated by a single ip injection of a mixture of acepromazin (0.5 mg/kg), midazolam (0.25 mg/kg) and fentanyl (0.025 mg/kg). Sedated animals were scanned in toto using a small animal DEXA scanner (pDEXA, Norland Stratec Medizintechinik GmbH, Birkenfeld, Germany) and data were analyzed by the software supplied by the manufacturer. Fat mass and lean body mass were determined.

45.4 30.1 18.3 24.1 35.1 21.4

43.6 1.8 30.1 18.3 23.1 1.0 35.1 21.4

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For statistical analysis, SPSS version 16.1 was used. The MannWhitney nonparametric test for 2 independent samples was used to dene differences between the groups of mice. The criterion for signicance was set at p < 0.05.

3. Results 3.1. Body weight and energy expenditure During the 12 weeks of high-fat diet intervention, the control mice gained 8.5 3.1 g of body weight (Fig. 1). PSO fed mice only increased 5.7 2.9 g, p = 0.03. DEXA analysis showed indeed a reduced fat mass in the PSO fed animals: 3.3 2.3 vs 6.7 2.7 g respectively p = 0.02 (Fig. 1). Analyses of energy expenditure using indirect calorimetry showed no signicant differences in total energy expenditure and food intake between the PSO and HFD only fed mice (Fig. 2 A and D). Interestingly, respiratory exchange ratio (carbohydrate and fat oxidation) of the PSO fed animals showed an increased RER indicative for relative elevated carbohydrate oxidation and decreased fat oxidation (Fig. 2 B). These shifts in substrate use suggest improvement of substrate exibility in PSO fed animals. To investigate this we performed a fasting and re-feeding experiment in the metabolic cage setup. After a 12 h fast PSO and HFD-only fed animals had similar low RER values indicative for increased fat oxidation. When re-fed, the PSO fed mice increased their RER more rapidly and to higher levels than the HFD-only fed animals, delta

Fig. 2. Energy expenditure assessed over a period of 24 h; average total energy expenditure (A); respiratore exchange rate (B) and accumulated food intake (C) for HFD (black dots) and PSO (white dots) fed mice. The night period is indicated by the gray area. Values represent mean SD for n = 8 vs 6 respectively.

RER 0.18 0.03 vs 0.13 0.03 for PSO vs HFD control animals, p < 0.05. These results imply that the PSO mice have an improved metabolic exibility. This improved metabolic exibility suggests improved insulin sensitivity in both mice as well as humans (Storlien et al., 2004).

3.2. Hyperinsulinemic euglycemic clamp analyses To investigate whether the PSO fed mice indeed have improved insulin sensitivity a hyperinsulinemic euglycemic clamp was performed. There were no signicant differences in fasted glucose or insulin levels between the two groups; glucose 4.5 0.7 mM vs 4.7 0.7 mM and insulin 0.4 0.2 ng/mL vs 0.4 0.1 ng/mL for HFD-only and PSO respectively. After the clamp we achieved euglycemia and equal insulin concentration in both groups (Table 2). The glucose infusion rate to maintain euglycemia during the hyperinsulinemic period was not signicantly different in the PSO fed group in comparison with HFD (Table 2, Fig. 3 A and B). Tracer analysis (Fig. 4) however, showed that PSO feeding led to higher stimulation of insulin-mediated peripheral (mainly muscle tissue (DeFronzo et al., 1981) glucose usage compared to HFD-only fed mice, 164 52% vs 92 24%, p = 0.01, (Fig. 4A) showing an increased whole body insulin sensitivity. Suppression of endogenous

Fig. 1. Body weight measured throughout the duration of the study at week 0, 6 and 12 of HFD (black bars) and HFD + PSO (white bars) feeding (A). DEXA body analysis performed at the end of the study (B). Values represent mean SD and indicates statistical signicant difference, Mann Whitney U-test p < 0.05.

I.O.C.M. Vroegrijk et al. / Food and Chemical Toxicology 49 (2011) 14261430 Table 2 Glucose infusion rate, plasma glucose and insulin concentrations at the end of the hyperinsulinemic euglycemic clamp analysis. Mice were put on a high-fat diet with or without PSO for 12 weeks. At the end of the diet intervention period the clamp analysis was performed, after an overnight fast. The values represent mean SD for n = 8 and 6 per group respectively. Control (n = 8) Glucose infusion rate (lmol/kg/min) Glucose (mmol/L) Insulin (ng/mL) Values represent mean SD. 30 4 4.8 0.8 4.9 1.4 PSO (n = 6) 37 10 4.8 1.0 4.2 1.2

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Fig. 4. Tracer analysis of the hyperinsulinemic euglycemic clamp. Insulin stimulated the whole body glucose utilization, BGU (%stimulation) and inhibition of endogenous glucose production EGP (hepatic glucose production) (%inhibition) in HFD (black bars) and PSO (white bars) fed mice respectively. Values represent mean SD for n = 8 vs 6 mice respectively, p < 0.05.

Fig. 3. Glucose concentration (A) and glucose infusion rates (B) for HFD (black dots) and PSO (white dots) fed mice during the hyperinsulinemic euglycemic clamp period. Values represent mean SD for n = 8 vs 6 mice respectively.

glucose production was not signicantly affected in the PSO fed group, indicating equal liver insulin sensitivity (Fig. 4B). 4. Discussion In this study the effect of Pomegranate seed oil, punicic acid on high fat diet-induced obesity and insulin resistance was investigated. Feeding a high fat diet in C57Bl/J6 mice leads to the induction of increased body fat mass as well as the induction of insulin resistance in several tissues. One gram of the fat in the high fat diet Palm oil was replaced with Pomegranate seed oil (PSO). Over a 12 week period the PSO fed animals gained less weight. C57Bl/J6 mice on a low fat or chow diet weight about 28.6 1.8 g, showing an increase of 3.2 1.2 g in 12 weeks. These data show that the PSO fed animals are protected against HFD-induced obesity in absolute sense. Our ndings conrm the data previously seen

in the CD1 mouse strain (McFarlin et al., 2009). Here it was also found that the decreased body weight was reected in reduced fat mass at the end of the 12 weeks feeding period. We did not nd a lower fasted glucose or insulin concentration in the PSO fed animals while McFarlin et al. (2009) describe a lower fasted insulin concentration. The use of a different mouse strain and a different high fat diet (amount and content) might in part explain this discrepancy (McFarlin et al., 2009). Food intake is a large determinant of body weight maintenance/gain. Analysis of daily food intake over a 24 h period did not show any difference between the PSO fed animals and the HFD-only fed controls. So alterations in energy intake via dietary means did not explain the decrease in body weight and fat mass. These data are in line with the observations made previously (McFarlin et al., 2009). Apart from the energy intake energy output was analyzed by indirect calorimetry. Daily total energy expenditure was not signicantly different between the groups. When the different macronutrient contribution to the energy expenditure was assessed by analyzing the RER an increased carbohydrate and a reduced fat contribution was observed. Since it is not possible to collect urine during our indirect calorimetry set up, we have to assume protein oxidation is unaltered. The equal lean mass of the PSO group compared to the HFD-only group in fact indicates that there is probably no alteration in protein oxidation induced by the PSO supplementation. The data for the energy expenditure is shown as kcal per hour per total mouse because of the difference in total body weight. Correction for lean mass did not alter the results and/or conclusions since there is no difference in lean mass between the two groups. So overall these data do not explain why there is a signicant lower body weight and fat mass gain in the PSO and HFD-control fed mice. We cannot exclude that more acute differences in energy balance induced by PSO during the rst 2 weeks of the dietary intervention might have induced the onset of a negative energy balance and that this might be normalized after 12 weeks. To study whether the reduction of obesity in PSO fed mice resulted in altered insulin sensitivity and glucose handling, we rst performed a fasting- re-feeding experiment in the indirect calorimetry cages to assess the metabolic exibility. The metabolic exibility is used to evaluate the capacity of an animal to respond to a nutrient switch. Metabolic exibility is determined as the response from fasting (full fat oxidation) to re-feeding (carbohydrate oxidation) (Storlien et al.,

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2004) and is indicative for insulin sensitivity. The PSO fed animals had a higher RER, indicative for a better response after re-feeding. This increased metabolic exibility suggests that the PSO-fed mice have increased insulin sensitivity (Storlien et al., 2004). To assess true insulin sensitivity, a hyperinsulinemic euglycemic clamp was performed next. Indeed PSO fed animals had higher insulin stimulated whole body glucose usage while the inhibition of endogenous glucose production was equal for both dietary groups. This suggests that hepatic (endogenous) insulin sensitivity is similar for the two groups while the periphery, mainly muscle tissue (DeFronzo et al., 1981), of the PSO fed mice is more insulin sensitive. The Palm oil based diet which was used as a reference did cause insulin resistance since the insulin stimulated glucose usage increased 100% in HFD-only mice while under low fat conditions the whole body glucose usage increases $300 24% (Goudriaan et al., 2005; Duivenvoorden et al., 2005; Voshol et al., 2001, 2003). As shown previously a palm oil enriched high fat diet causes insulin resistance in muscle but not in liver (van den Berg et al., 2010b). We are the rst to show that PSO supplementation indeed increases peripheral insulin sensitivity in mice. Previous data by McFarlin et al. (2009) indicate that insulin sensitivity is improved in their study based on the lower insulin concentration without true glucose metabolism measurement. Our data conclusively show that PSO supplementation improves insulin sensitivity in HFD-fed mice and conrm at least in part the ameliorated glucose tolerance as previously found (Hontecillas et al., 2009). Further research is needed to investigate the underlying mechanisms but the stimulation of PPARy by PSO is one of the possible mechanisms as shown previously (Hontecillas et al., 2009). These observation are in line with ndings using other CLA-like fatty acids (MoyaCamarena et al., 1999; Bassaganya-Riera et al., 2004; Hontecillas et al., 2008) making PSO a useful supplementation. The anticipated human dose is 3 g PSO/day ($50 mg/kg body weight/day), either by supplementation or pomegranate fruit. Giving species differences this would equal $500 mg PSO/kg body weight/day in mice. In the current study we used a dose of 1 g PSO/kg body weight/day with the described ndings. We conclude that supplementation with Pomegranate seed oil in mice protects against HFD-induced weight gain and fat mass gain, e.g. obesity and insulin resistance.

the Nutrigenomics Consortium, the seventh framework program of the EU-funded LipidomicNet (Proposal number 202272). References
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5. Conict of Interest The authors declare that there are no conicts of interest. Acknowledgements This work was performed in the framework of the Leiden Center for Cardiovascular Research LUMC-TNO and supported by grants from the Netherlands Organization for Scientic Research (NWO Zon-MW; 917.76.301 to Peter J. Voshol) and the Dutch Diabetes Research Foundation (2005.01.003 to Peter J. Voshol), SenterNovem Grant IS055036, the Centre for Medical Systems Biology (CMSB) in the framework of the Netherlands Genomics Initiative (NGI) (Project 115), Top Institute Pharma (Project T2-105) and