Even Fjære Effects of macronutrient composition and cyclooxygenase-inhibition on diet-induced obesity, low grade inflammation and glucose homeostasis

Background: Polyunsaturated n-3 fatty acids (n-3 PUFAs) are reported to protect against high fat diet-induced obesity and inflammation in adipose tissue. Here we aimed to investigate if the amount of sucrose in the background diet influences the ability of n-3 PUFAs to protect against diet-induced obesity, adipose tissue inflammation and glucose intolerance. Methodology/Principal Findings: We fed C57BL/6J mice a protein(casein) or sucrose-based high fat diet supplemented with fish oil or corn oil for 9 weeks. Irrespective of the fatty acid source, mice fed diets rich in sucrose became obese whereas mice fed high protein diets remained lean. Inclusion of sucrose in the diet also counteracted the well-known antiinflammatory effect of fish oil in adipose tissue, but did not impair the ability of fish oil to prevent accumulation of fat in the liver. Calculation of HOMA-IR indicated that mice fed high levels of proteins remained insulin sensitive, whereas insulin sensitivity was reduced in the obese mice fed sucrose irrespectively of the fat source. We show that a high fat diet decreased glucose tolerance in the mice independently of both obesity and dietary levels of n-3 PUFAs and sucrose. Of note, increasing the protein:sucrose ratio in high fat diets decreased energy efficiency irrespective of fat source. This was accompanied by increased expression of Ppargc1a (peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha) and increased gluconeogenesis in the fed state. Conclusions/Significance: The background diet influence the ability of n-3 PUFAs to protect against development of obesity, glucose intolerance and adipose tissue inflammation. High levels of dietary sucrose counteract the antiinflammatory effect of fish oil in adipose tissue and increases obesity development in mice. Citation: Ma T, Liaset B, Hao Q, Petersen RK, Fjære E, et al. (2011) Sucrose Counteracts the Anti-Inflammatory Effect of Fish Oil in Adipose Tissue and Increases Obesity Development in Mice. PLoS ONE 6(6): e21647. doi:10.1371/journal.pone.0021647 Editor: Aimin Xu, University of Hong Kong, China Received March 28, 2011; Accepted June 4, 2011; Published June 28, 2011 Copyright: 2011 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Danish Natural Science Research Council, the Novo Nordisk Foundation, the Carlsberg Foundation, and the National Institute of Nutrition and Seafood Research, Norway. Part of the work was carried out as a part of the research program of the Danish Obesity Research Centre (DanORC). DanORC is supported by the Danish Council for Strategic Research (Grant NO 2101 06 0005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (KK); [email protected] (LM) ¤ Current address: Department of Food Safety and Nutrition, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway Introduction Today it is recognized that the potentially harmful effects of high fat diets relates to not only the amount, but also the type of dietary fatty acids. Whereas a high intake of saturated and trans fatty acids has been shown to be associated with increased risk of cardiovascular diseases in several studies, intake of polyunsaturated fatty acids (PUFAs) has been associated with lower cardiovascular risk [1,2]. Thus, increasing the relative amount of PUFAs, both vegetable n-6 PUFAs and marine n-3 PUFA, at the expense of saturated fat is recommended. It is important to note, however, that more than 85% of the total dietary PUFA intake in Western diets today is vegetable n-6 PUFAs, mainly linoleic acid [3]. This is largely due to the high amount of linoleic acid in corn-, sunflower-, and soybean-oil used in both home-cooking and in industrially prepared food [4]. Moreover, animal feeds are enriched with n-6 PUFAs, and although meat production methods are diverse, meat fatty acid profiles will always reflect that of the animal feed [4]. Thus, the dietary n-3:n-6 PUFA ratio has decreased [3,4]. Although the exchange of saturated fat with vegetable n-6 PUFAs may have some beneficial effects on human health, a low n-3:n-6 PUFAs ratio is associated with a high risk of several lipid-related disorders [2,3]. A high intake of n-6 PUFAs has also been associated with childhood obesity, [4,5]. Animal studies have shown that feeding mice a diet containing the n-6 PUFA, linoleic acid, during the pregnancy-lactation period leads to obesity in the offspring [6]. This effect, however, is prevented by inclusion of the n-3 PUFA a-linolenic acid in the diet [6]. These findings are in line with several studies demonstrating that dietary n-3 PUFAs are able to limit the development of diet-induced obesity [7–13]. PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21647 Obesity may be considered as a state of chronic low-grade inflammation [14,15]. Accumulated evidence strongly suggests that low grade chronic inflammation plays a crucial role in development of obesity related insulin resistance [16]. Furthermore, it should be noted that continuous subcutaneous infusion of lipopolysaccharide (LPS) is sufficient to induce adipose tissue inflammation, insulin resistance and obesity in mice [17]. It is also well documented that n-3 PUFAs are able to limit high fat diet-induced inflammation in adipose tissue in rodents [18–20]. Both n-3 PUFAs and n-6 PUFAs are substrats for cycloand lipoxygenases and n-3 PUFAs are traditionally assumed to act anti-innflammatory by competitive inhibition of the biosynthesis of arachidonic acid-derived pro-inflammatory prostaglandins of the 2-series and furthermore, n-3 PUFA-derived prostaglandins of the 3-series are believed to be less inflammatory [21,22]. Recent research furthermore demonstrate that n-3 PUFAs may be converted to anti-inflammatory cyclooxygenase-2 derived electrophilic oxoderivatives and resolvins [21,22]. Moreover, by activation of the fatty acid receptor, GPR120, n-3 PUFAs repress LPSand TNFamediated inflammatory signalling responses, and thereby increase insulin sensitivity by repressing macrophage-induced adipose tissue inflammation [23]. Thus, consumption of n-6 PUFAs at the expense of n-3 PUFAs may aggravate the metabolic consequences of obesity. Increasing the dietary intake of n-3 PUFAs is therefore currently recommended by several health authorities. In order to curb the increasing obesity problem, nutritionists and authorities have largely focused on reducing fat intake, as dietary fat contains more energy per gram than proteins and carbohydrates. As an alternative to low energy diets, low carbohydrate diets are becoming increasingly popular although still controversial. The mechanisms by which such diets induce weigh loss are still not fully elucidated, but it has been documented that high protein diets increase energy expenditure in part due to a thermic effect [24]. We have previously shown that the protein:sucrose ratio in the background diet determines the adipogenic potential of dietary n-6 PUFAs in mice [25]. Mice fed n-6 PUFAs in combination with sucrose became obese, and had a markedly higher feed efficiency than mice pair-fed n-6 PUFAs in combination with proteins [25]. In fact, the high-protein fed mice needed almost 7 times more energy to achieve a weight gain of 1 g than mice on the high-sucrose diet [25]. The high protein diet led to an increased glucagon/insulin ratio, concomitant with elevated cAMP-dependent signaling, induction of COX-mediated prostaglandin synthesis and increased expression of uncoupling protein-1 (UCP1) in inguinal subcutaneous white fat [25]. In the present paper we aimed to investigate whether this phenomenon is restricted to n-6 PUFAs or if the effects of dietary fats, such as fish oils, which are considered beneficial to human health, also depend on the background diets. Furthermore, we aimed to examine whether the background diet exerts an influence on the ability of n-3 PUFAs to protect against glucose intolerance and adipose tissue inflammation. Results Sucrose counteracts the obesity-reducing effect of fish oil in ad libitum fed mice It is a general notion that intake of fish oil rich in n-3 PUFAs limits high fat diet-induced obesity in rodents, whereas diets rich in n-6 PUFAs have been associated with an increased propensity to develop obesity [6,26]. As we have demonstrated that the obesogenic effect of n-6 PUFAs is determined by the content of carbohydrates and protein in the feed [25], we speculated whether the effect of dietary fats considered health-beneficial, such as fish oil, might be modulated by different background diets. To answer this question we fed C57BL/6J male mice isocaloric high fat diets (Table 1 and 2) containing corn oil or fish oil supplemented with either protein or sucrose or a conventional low fat diet ad libitum for 9 weeks. Contrasting the general notion that fish oil attenuates high fat diet-induced obesity, the mice fed the fish oil in combination with sucrose gained as much body weight as the mice fed corn oil and sucrose (Fig. 1A and B). When combined with sucrose, fish oil did not reduce the weights of neither epididymal (eWAT) nor inguinal white adipose tissue (iWAT) mass compared with corn oil (Fig. 1E and F). Moreover, morphological analyses demonstrated that the adipocyte size was similar in the two sucrose fed groups (Fig. 1G). Of note, weight gain in mice fed corn oil or fish oil plus protein were indistinguishable from that of mice fed the low fat diet (Fig. 1A). Compared with low fat fed mice, the weights and adipocytes sizes of eWAT and iWAT in mice fed both high fat diets in combination with protein tended to be smaller, but the differences did not reach statistical significance (Fig. 1E, F and G). Thus, when combined with a high intake of sucrose fish oil did not prevent obesity. However, high dietary protein content prevented weight gain and obesity when combined with either corn or fish oil. Sucrose, but not protein or fat, strongly stimulates pancreatic insulin secretion, and accordingly, plasma levels of insulin were consistently higher in mice fed the sucrose-based diets than in mice fed the protein-based diets (Fig. 1C). Conversely, the levels of plasma glucagon were lower and hence, the insulin:glucagon ratio was about three times higher in mice fed high sucrose than in mice fed high protein irrespective of whether the diets contained corn oil or fish oil (Fig. 1D). Collectively, these results indicate that intake of sucrose and hence increased insulin secretion, abrogates the protective effects of fish oil in relation to adipocyte hyperplasia and hypertrophy and thereby the development obesity. Sucrose counteracts the anti-inflammatory effect of fish oil in adipose tissue The ability of n-3 PUFAs to limit high fat diet-induced inflammation in adipose tissue is well documented [18–20]. As chronic low grade inflammation in adipose tissue is a characteristic trait of obesity [14,15] and sucrose abrogates the anti-adipogenic effect of fish oil, we asked whether the background diet also attenuated the ability of n-3 PUFAs to protect against adipose tissue inflammation. Gene expression analyses of eWAT and iWAT revealed a striking correlation between macrophageand inflammatory markers and the intake of sucrose-based diets irrespectively of the fat source (Fig. 2A). Expressions of macrophage marker genes Emr1 (EGF-like module containing, mucin-like, hormone receptor-like sequence 1 or F4/80) and Cd68, as well as markers of inflammation Serpine1 (Plasminogen activator inhibitor1) and Ccl2 (chemokine (C-C motif) ligand 2), were significantly higher in adipose tissue from mice fed sucrose than in mice fed high protein or a low fat diets (Fig. 2A). Moreover, we noticed a significant increase in the expression of Pparg (peroxisome proliferator-activated receptor c) in eWAT in the mice fed protein supplemented with corn oil compared with mice fed protein supplemented with fish oil (Fig. 2A). A high fat diet impairs glucose tolerance independent of macronutrient composition and obesity As adipose tissue inflammation is causally linked to development of insulin resistance and glucose intolerance, we subjected mice fed the different diets for 9 weeks to an intraperitoneal glucose tolerance test (GTT). Surprisingly, the GTT demonstrated that Fish Oil, Sucrose and Obesity PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21647 the glucose tolerance was impaired both in the mice fed the protein-based diets and in the mice fed the sucrose-based diets (Fig. 2B). Evidently, impaired glucose tolerance was dissociated from the state of obesity, suggesting that intake of relatively high amounts of fat reduces glucose tolerance even if weight gain and expression of inflammatory markers were maintained at low levels. However, fasting glucose and insulin levels were lower in mice fed high protein than high sucrose. Thus, calculation of HOMA-IR indicated that the mice fed proteins remained insulin sensitive, whereas insulin sensitivity tended to be reduced in the obese mice fed sucrose even though the difference between sucrose and protein fed mice did not reach statistical significance (Fig. 2B). Sucrose does not reduce the ability of fish oil to prevent diet-induced accumulation of fat in the liver As the anti-inflammatory effect of n-3 PUFAs in adipose tissue is well documented, we investigated if the high level of dietary sucrose reduced uptake of n-3 PUFAs. Thus, GC-MS analyses were performed to determine the fatty acid composition in red blood cells, liver and adipose tissues. These analyses demonstrated the expected enrichment of n-3 PUFA in lipids in red blood cells and liver (Table 3 and 4). In adipose tissue, the enrichment of n-3 was actually higher in mice fed the sucrose-based fish oil diet than in the protein-based fish oil diet group (Table 4). However, inclusion of sucrose in the diet did not reduce the ability of fish oil to prevent accumulation of fat in the liver (Fig. 3A). When sucrose was included in the diet, lipid accumulation in livers from fish oil fed mice was significantly lower than in livers from mice fed corn oil (Fig. 3A). Moreover, expressions of lipogenic genes seem to be determined by the sucrose: protein ratio independent of fat source (Fig. 3B). Thus, the ability of fish oil, but not corn oil, to protect against diet-induced lipid accumulation in the liver did not seem to be directly related to the suppression of lipogenic gene expression. Other hallmarks of n-3 PUFA actions are their ability to increase fatty acid oxidation and to reduce plasma triacylglycerol levels [27,28]. Plasma triacylglycerol levels were significantly reduced in mice fed fish oil in combination with proteins, but inclusion of sucrose abrogated this effect (Fig. 3C). The higher plasma levels of b-hydroxybutyrate in mice fed fish oil in combination with proteins indicated that hepatic fatty acid oxidation was increased in these mice, and inclusion of sucrose attenuated this effect (Fig. 3C). Together these results demonstrate that the protein:sucrose ratio also affects the ability of fish oil to reduce plasma levels of triacylglycerol and increase fatty acid oxidation. Increasing the protein:sucrose ratio in a high fat diet decreases energy efficiency irrespective of corn or fish oil supplementation To verify that the obesity in mice fed fish oil in combination with sucrose was simply not due to increased energy-intake, feed intake was recorded and energy efficiency calculated. Obviously, energy intake was significantly higher in mice fed high fat diets than that of mice receiving the low fat diet (Fig. 3D). Energy intake tended to be higher in mice receiving the sucrose diets than in mice fed the protein-based diets, but this was not statistically significant (Fig. 3D). Thus, energy efficiency was dramatically increased in mice receiving sucrose compared to protein, indicating difference in energy expenditure. A simple way to detect differences in catabolic rate is to subject mice to fasting and measure the resulting weight loss. Figure 3D shows that mice on the protein-based diets lost significantly more weight during 18 h of fasting. This supports the notion that energy expenditure is higher in mice on a protein-based diet irrespective of whether the diet is supplemented with corn oil or fish oil. Expression and activation of UCP1 in brown and white adipose tissue lead to dissipation of energy in the form of heat, and may thus protect against diet induced obesity [29]. Gene expression analyses of adipose tissues demonstrated that expression of Ucp1 (uncoupling protein-1), in mice fed high protein was higher in iWAT, but not in eWAT or (iBAT) (Fig. 3E). Increased expression of Ucp1 in iWAT in mice fed the protein-based diets was accompanied by increased expression of Cpt1b (carnitine palmitoyltransferase-1b), Ppargc1a (peroxisome proliferator-activated receptor gamma coactivator 1 alpha) and Dio2 (deiodinase, iodothyronine, type II), suggesting that iWAT adopted a more brown-like phenotype (Fig. 3E). Thus, the lean phenotype in mice fed the high protein diets, appears, at least in part, to result from increased uncoupled respiration in iWAT. The obesogenic effect of fish oil is determined by the macronutrient composition in pair-fed mice Since energy intake was slightly higher in mice receiving fish oil in combination with sucrose compared with protein, we decided to demonstrate directly that this difference was insufficient to account for the increased adipose tissue mass. Accordingly, mice were pairfed the isocaloric diets containing fish oil in combination with sucrose or protein. To achieve identical energy intake we recorded Table 1. Macronutrient composition in the diets. High fish oil High corn oil Low energy Sucrose Protein Sucrose Protein Protein (g/kg) 200 200 540 200 540 Casein 200 200 540 200 540 L-Cysteine 3 3 3 3 3 Carbohydrate (g/kg) 619.5 439.5 99.5 439.5 99.5 Corn starch 529.5 9.5 9.5 9.5 9.5 Sucrose 90 430 90 430 90 Fat (g/kg) 70 250 250 250 250 Soybean oil 70 70 70 70 70 Corn oil 180 180 Fish oil 180 180 doi:10.1371/journal.pone.0021647.t001 Table 2. Fatty acid composition in the diets. High fish oil High corn oil Low energy Sucrose Protein Sucrose Protein SFA (mg/g) 9.6 53.3 50.3 31.4 31.4 MUFA (mg/g) 17.0 54.3 54.0 65.9 65.9 PUFA (mg/g) 36.2 100.6 99.5 120.8 121.2 n-6 (mg/g) 32.7 40.6 38.9 117.3 117.8 n-3 (mg/g) 3.5 60.0 60.6 3.5 3.4 n-3/n-6 0.11 1.48 1.56 0.03 0.03 Abbreviations: SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. doi:10.1371/journal.pone.0021647.t002 Fish Oil, Sucrose and Obesity PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21647 the ad libitum feed intake of mice receiving the protein-based diet, and restricted the amount of feed to mice receiving the sucrosebased feed accordingly. Figure 4A demonstrates that even under conditions of pair-feeding, the mice receiving high sucrose gained dramatically more weight than those receiving a high protein diet. Similarly, as observed in ad libitum fed mice, energy efficiency and adipose tissue mass were significantly higher when mice were fed sucrose (Fig. 4B and C). Moreover, energy content in the feces was similar in both groups and the apparent digestibility was not increased by increased sucrose amount in the diet (Fig. 4B). Plasma levels of insulin were higher and glucagon lower in mice fed the sucrose than protein (Fig. 4D). In iWAT, but not eWAT, we observed a significant induction of Ppargc1a and Ucp1 expression indicative of the transformation of iWAT into a more brown-like depot in protein fed mice (Fig. 4E). In iBAT, expressions of Ucp1 and cyt COXII, (cytochrome c oxidase, subunit II) a marker of mitochondrial content, were not significantly different in mice fed protein or sucrose (Fig. 4F). Lower expression levels of inflammatory markers in eWAT and iWAT in protein fed mice were also confirmed (Fig. 4E). In addition, as observed in ad libitum fed mice, glucose tolerance was similarly affected in protein and sucrose fed mice (Fig. 5A). Fasting levels of insulin were higher in sucrose+fish oil fed mice than the two other groups, but an ITT test showed no significant difference between the groups (Supp Fig. 1). Plasma levels of triacylglycerol were lower and b-hydroxybutyrate were higher in plasma from mice fed fish oil in combination with protein than in mice fed fish oil in combination with sucrose (Fig. 5B). However, expression of genes involved in fatty acid oxidation was not increased in liver (Fig. 6B) or in muscle (not shown). Actually, expression of the classical PPARa target Acox1 (acyl-CoA oxidase 1) was higher in liver of sucrose fed mice (Fig. 6B). Thus, a possible increase in fatty acid oxidation in protein fed mice as indicated by the elevated levels of b-hydroxybutyrate did not appear to be due to increased expression of genes involved in fatty acid oxidation. Of note, however, higher expressions of Srebf1 (sterol regulatory element binding transcription factor 1) as well as Acaca (acetyl-Coenzyme A carboxylase alpha) and Fasn (fatty acid synthase), indicate that sucrose overrides the suppressive effect of fish oil on lipogenic gene expression. Energy expenditure is reduced in mice fed fish oil in combination with sucrose Mice fed fish oil in combination with sucrose exhibited increased weight gain and an increased feed efficiency indicative of decreased energy expenditure. Moreover, mice fed fish oil in combination with sucrose lost significantly less weight during 18 h of fasting (Fig. 3C). Therefore, we examined whether energy Figure 1. Sucrose counteracts the obesity-reducing effect of fish oil in ad libitum fed mice. Male C57BL/6 mice (n = 8) were fed isocaloric high fish oil or high corn oil diets with different carbohydrate and protein contents ad libitum for 9 weeks. A: Body weight development of ad libitium fed mice. (B) Prior to termination the mice were photographed. C–D: Insulin and glucagon levels were measured in plasma in the fed state. E–G: The weights of epididymal and inguinal white adipose tissues were recorded and sections were stained with hematoxylin and eosin. Data are presented as means 6 SEM. Different small letters denote significant differences between the groups (P,0.05). doi:10.1371/journal.pone.0021647.g001 Fish Oil, Sucrose and Obesity PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21647 expenditure was reduced when fish oil was combined with sucrose. Accordingly, O2 consumption and CO2 production were measured by indirect calorimetry. Figure 5C shows that O2 consumption both in the light and the dark periods tended to be lower in mice fed fish oil in combination with sucrose than with protein. As expected, mice fed fish oil in combination with sucrose had a higher CO2 production resulting in a statistically significant higher RER of about 0.9 indicating a lower rate of fatty acid oxidation (Fig. 5C). A diet enriched with fish oil and proteins increases gluconeogenesis High circulating levels of insulin combined with a low level of glucagon translate into reduced cAMP signalling in the liver. Thus, the observed reduced expressions of Crem (cAMP responsive element modulator), Pde4c (phosphodiesterase 4C, cAMP specific), Ppargc1a and Pck1 (phosphoenolpyruvate carboxykinase 1, cytosolic) as well as reduced expressions of enzymes involved in amino acid degradation in the liver of sucrose fed mice were anticipated (Fig. 4D). In the liver PGC1a is induced in response to elevated levels of cAMP and plays a central role in the control of hepatic gluconeogenesis [30–32]. In keeping with the increased expressions of Ppargc1a and Pck1 in liver from mice fed fish oil in combination with protein compared to sucrose, we anticipated that gluconeogenesis was induced in the fed state in the protein fed mice. To measure gluconeogenesis in vivo mice fed fish oil in combination with either protein or sucrose were intraperitoneally injected with pyruvate both after overnight fasting and in the fed Figure 2. A high fat diet impairs glucose tolerance independent of macronutrient composition and obesity. A: Expressions of adipogenic and inflammatory marker genes (Pparg (peroxisome proliferator activated receptor c), Adipoq (adiponectin), Serpine1 (Plasminogen activator inhibitor-1), Ccl2 (chemokine (C-C motif) ligand 2), Emr1 (EGF-like module containing, mucin-like, hormone receptor-like sequence 1 or F4/ 80) and Cd68 (CD68 antigen)) were measured in epididymal and inguinal white adipose tissue using RT-qPCR (n = 8). B: Intraperitoneal glucose tolerance test was performed in a separate set of mice (n = 10). Fasting glucose and insulin levels were measured to calculate HOMA-IR. Data are presented as means 6 SEM. Different small letters denote significant differences between the groups, in 2A within the same tissue (P,0.05). doi:10.1371/journal.pone.0021647.g002 Fish Oil, Sucrose and Obesity PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21647 state, and blood glucose was measured in the following 60 minutes. In the fasted state, mice fed sucrose or protein exhibited similar excursions, indicating similar rates of gluconeogenesis (Fig. 6A). In fed mice, however, the rise of blood glucose following the injection of pyruvate was dramatically faster and reached much higher levels after 15 and 30 min in the protein fed mice than in chow fed mice (Fig. 6A). Compared with chow fed mice the rise in blood glucose was also increased in mice fed fish oil in combination with sucrose, but this was not statistically significant (Fig. 6A). The decline in blood glucose in chow fed mice remains to be explained, but this was observed consistently. Taken together these results strongly support the assumption that gluconeogenesis is markedly induced in mice fed the protein-based diet. Discussion It is well documented that inclusion of n-3 PUFAs in high fat diets leads to reduced development of diet-induced obesity in rodents [7–9,11–13,33]. Unfortunately, not all studies where the anti-obesogenic effects of fish oils are studied provide a detailed description of the macronutrient composition. However, in standard commercial available high fatand very high fat diets, starch is the most abounded carbohydrate source and the amount of sucrose is low or absent. Here we show that a high amount of sucrose in the diet counteracts the obesity-reducing effect of fish oil as well as the well described anti-inflammatory effect in adipose tissue [19,23,34,35]. Irrespective of the fatty acid source, mice fed high protein diets remained lean whereas mice fed diets enriched in sucrose became obese and had higher expressions of inflammatory markers in adipose tissue. Collectively, our results demonstrate that a high intake of sucrose abrogates the protective effects of fish oil in development of obesity. As dietary sucrose, but not protein or fat, stimulates secretion of insulin from pancreatic b-cells, an increased dietary sucrose:protein ratio will translate into an increased insulin:glucagon ratio in the fed state. In this respect the observed higher insulin:glucagon ratio in mice fed the sucrose-based diets than in mice fed the protein-based diets was expected. Increased levels of insulin in fed mice were observed irrespectively of the type of fat in the diet. Insulin is a powerful anabolic hormone that stimulates adipocyte differentiation and adipose tissue expansion [36]. Activation of insulin signaling is crucial for the development of obesity [37] and insulin receptor substrate-1 (IRS-1) transgenic mice are obese [38]. Increased insulin signaling and glucose uptake in adipose tissue in the fed state in sucrose fed mice may thus override the protective effect of fish oil when it comes to protection against Table 3. Fatty acid composition in red blood cells. High fish oil High corn oil Low energy Sucrose Protein Sucrose Protein Sum total (mg/g) 3.41±0.04 3.04±0.07 2.87±0.05 3.52±0.11 3.34±0.10 SFA (mg/g) 1.28±0.02 1.24±0.03 1.17±0.02 1.31±0.05 1.21±0.04 MUFA (mg/g) 0.49±0.01 0.34±0.01 0.35±0.01 0.41±0.01 0.40±0.01 PUFA (mg/g) 1.39±0.02 1.28±0.03 1.19±0.02 1.53±0.05 1.44±0.04 n-3 (mg/g) 0.2560.00 0.7960.02 0.7360.01 0.2160.01 0.1160.00 n-6 (mg/g) 1.1460.02 0.4960.01 0.4760.01 1.3260.05 1.3460.04 n-3/n-6 0.2260.00 1.6260.01 1.5560.28 0.1660.00 0.0860.00 Data are presented as mean 6 SEM (n = 8). doi:10.1371/journal.pone.0021647.t003 Table 4. Fatty acid composition in organs. High fish oil High corn oil Low energy Sucrose Protein Sucrose Protein Liver Sum total (mg/g) 45±4 41±1 35±2 71±10 42±3 % 100 100 100 100 100 SFA (mg/g) 15±1 15±0 12±1 21±3 13±1 % 32.560.9 36.560.4 35.260.3 29.660.3 31.360.8 MUFA (mg/g) 13±2 6±1 4±0 19±4 7±1 % 29.161.6 14.962.3 11.960.7 25.661.9 17.261.3 PUFA (mg/g) 16±1 19±1 18±1 29±3 20±1 % 36.761.6 47.262.0 51.260.4 42.761.9 47.860.9 n-3 (mg/g) 3.2460.27 12.9460.51 10.8360.72 3.2860.15 1.7260.11 % 7.2560.45 31.4161.25 31.3460.35 4.9760.53 4.1060.28 n-6 (mg/g) 1361 660 76