Abstract

Introduction

Dietary advice to replace saturated fat with PUFA to reduce the incidence of CVD^(1, 2), and increasing use of vegetable oils, have led to a dramatic increase in the human consumption of linoleic acid (LA, 18:2n-6) during the twentieth century⁽³⁾. The estimated per capita consumption of soyabean oil, one of the major dietary sources of LA in the USA, has increased more than 1000-fold from 1909 to 1999, increasing the availability of LA from 2·8 to 7·2 % of energy (en%)⁽³⁾. Dietary intakes of n-3 andn-6 fatty acids are critical determinants of tissue proportions of bioactive 20- and 22-carbon n-3 andn-6 highly unsaturated fatty acids. The tissue fatty acid composition reflects dietary PUFA intake since 18-carbon n-3 and n-6 fatty acids cannot be synthesised de novo ⁽⁴⁾. The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) are endogenous lipid mediators formed from the pool of arachidonic acid (AA) in membrane phospholipids (AA-PL). Hence, endocannabinoid activity can be altered by dietary fatty acids that in turn affect endocannabinoid precursor levels after a short term^(5, 6) or prolonged feeding^(7 – 10). Dietary LA⁽¹⁰⁾ and AA^(5, 7, 11) increased, whereas dietary n-3 PUFA^{(5, 6, 10, 12 – 15)}, decreased 2-AG and AEA. The activation of the cannabinoid receptor 1 by endocannabinoids or exogenous agonists, centrally and peripherally, favours metabolic processes that stimulate appetite, increase food intake, activate fat storage pathways and down-regulate catabolism resulting in adipose accumulation^{(8, 10, 16)}.

The type of fat, specifically the imbalance in n-6 ton-3 PUFA, is emerging as a risk factor for developing obesity^{(17 – 20, 7)}. Dietary LA have been shown to have adipogenic properties in both humans⁽²¹⁾ and rodents^{(18, 19, 22 – 25)}. Fish oils rich in EPA and DHA limit diet-induced obesity in rodents^(26, 27) and are associated with weight reduction in humans^{(28 – 30)}. We have previously shown a robust positive correlation between the consumption of soyabean oil, the major contributor to dietary LA, and obesity in human subjects⁽¹⁰⁾. When investigating these human dietary changes over the last century in an animal model, we have demonstrated that it is the n-3 and n-6 fat composition of the diets, rather than the total amount of fat, that determined the obesogenic properties of a diet⁽¹⁰⁾. High-fat diets (60 en%) commonly used to induce obesity typically utilise soyabean oil and contain high levels of LA. Such diets elevate endocannabinoid levels in tissues involved in energy homeostasis contributing to diet-induced obesity in mice after long-term feeding^(7,8). We have recently reversed the obesogenic effect of high-fat isoenergetic diets by decreasing dietary LA from 8 to 1 en% and attenuated the AA-dependent excessive endocannabinoid activity⁽¹⁰⁾. Furthermore, we have demonstrated that reducing the AA-PL precursor pool by adding 1 en% EPA and DHA to 8 en% LA diets reversed both the stimulation of endocannabinoid activity and the obesogenic effect of high-fat diets⁽¹⁰⁾. Reducing excessive endocannabinoid system activity is actively being pursued to reduce obesity⁽⁸⁾. The pharmacological blockade of the cannabinoid receptor 1 is effective in treating obesity and related metabolic derangements^(31, 32). However, serious psychiatric side effects caused the withdrawal of rimonabant, a selective cannabinoid receptor 1 antagonist^(33, 34). Thus, there is a definite need for a dietary approach to reduce substrate availability for endocannabinoid synthesis.

The high degree of conservation of endocannabinoid system components⁽³⁵⁾ and the presence of the cannabinergic system in most animal systems^{(36 – 40)} point out the importance of the endocannabinoid system in the regulation of basic physiological responses such as energy homeostasis and feeding behaviour^{(36 – 38, 41, 42)}. Farmed Atlantic salmon have traditionally been fed diets based on fish oil and fishmeal, thus being recognised as a rich source of the marine n-3 fatty acids EPA and DHA. The steady increase in aquaculture production volume of 8–10 % per year⁽⁴³⁾ has resulted in the increased use of alternative proteins and oils in aqua feeds. Vegetable oils are recognised as suitable alternatives to fish oils^(44, 45), although they are devoid of EPA and DHA, with high levels of LA and monounsaturates, thereby reducing EPA and DHA and increasing LA content of fish fillet^{(44 – 46)}. The current fish oil replacement levels in aqua feeds are approximately 50 %, resulting in approximately 2 g EPA+DHA/100 g salmon flesh (www.nifes.no). This fish oil replacement level is expected to increase in future aquaculture feeds as Atlantic salmon production volumes increase⁽⁴³⁾whereas global availability of fish oil remains constant (www.iffo.net). However, there is a lack of documentation of the health consequences of replacing fish oil with vegetable oil in feed for Atlantic salmon in fish consumers. We here posit that excessive dietary LA elevates endocannabinoid activity in salmon and mice, with a concomitant increase in adiposity in mice fed LA-enriched salmon.

Methods

Feeding experiment in Atlantic salmon

 

Animals

Atlantic salmon (initial weight 340 (SEM 17) g (n 55)) were randomly distributed to six fibreglass tanks (1·5 × 1·5 × 0·9 m, water depth 0·6 m) provided with a continuous flow of seawater maintaining an average salinity of 35‰, an average temperature 8°C and a 12 h light–dark cycle. Mortality was recorded daily. The feeding trial was carried out at the Matre Aquaculture Research Station (Matredal, Norway; 60°52′N, 05°35′E) from 28 April 2008 to 6 October 2008.

 

Diets

Experimental diets (EWOS Innovation) provided as pellets were fed ad libitum to triplicate tanks per dietary treatment for 6 months. The diets contained the same 6 mm base pellet and were supplemented with either cleaned fish oil (FO; FF Skagen) or refined soyabean oil (SO; Mills) (Table 1) to ensure negligible amounts of contaminants such as persistent organic pollutants and polyaromatic hydrocarbons.

Table 1

Nutrient composition of mice and salmon diets

 

Sampling

Fish were feed-deprived 24 h before killing. From each tank, six fish were randomly sampled and killed with a sharp blow to the head to ensure no contamination of anaesthetics in the fish fillet. Body weight and length were measured, blood was collected and liver was quickly snap-frozen in liquid N₂ and stored at − 80°C until further analysis. The fillets were collected for use in mice feed.

 

Lipid extraction and fatty acid analysis

Total lipid was extracted from the Atlantic salmon diets, liver and fillets by homogenisation in chloroform–methanol (2:1, v/v) with 19:0 methyl ester as the internal standard. Fatty acid methyl esters (FAME) were prepared from total lipid by BF₃ following saponification, as described previously^(47, 48). Lipid classes of salmon liver were determined essentially as described by Jordal et al. ⁽⁴⁹⁾ based on Bell et al. ⁽⁵⁰⁾.

Effect of the experimental diets in Atlantic salmon

Replacing dietary FO with SO resulted in a 19-fold increase in LA, significantly higher AA levels and nearly doubled the concentration of 2-AG (Fig. 1) in the salmon liver. There was no difference in final body weight, mean visceral somatic index ((visceral adipose tissue+internal organ)/body weight) or whole-fish proximate composition after 6 months of feeding (Table 2). However, fish fed the SO diet had significantly higher amounts of TAG and total lipid in the liver than fish fed the FO diet, with no difference in PL (Table 2). Feeding SO to Atlantic salmon increased fillet LA (530 %) and reduced EPA (71 %) and DHA (56 %) compared with fillets from Atlantic salmon fed FO (Table 3).

Fig. 1

Levels of the n-6 fatty acids (a) linoleic acid (LA) and (b) arachidonic acid (AA), and the endocannabinoids (c) 2-arachidonoylglycerol (AG) and (d) anandamide (AEA) in the liver of Atlantic salmon fed soyabean oil (SO, ) and fish oil (FO, ). Values…

Table 2

Physical parameters in Atlantic salmon fed fish oil (FO) or soyabean oil (SO)

Table 3

n -6 and n-3 profile in total lipids of fillet and liver of Antlantic salmon fed fish oil (FO) and soyabean oil (SO)

Effect of the experimental diets in mice

Mice fed the SO salmon diet had higher amounts of LA and AA in the liver PL (Fig. 2(a) and (b)), erythrocyte-PL and neutral lipids of eWAT than mice fed the FO salmon diet (Table 4). Increasing dietary LA by replacing FO with SO in feed for Atlantic salmon significantly elevated liver 2-AG, elevated brain 2-AG and AEA, and significantly increased weight gain, feed efficiency and caused higher body weight (P < 0·08) in mice (Fig. 2). Mice fed the SO salmon diet had higher body weight than mice fed the FO salmon diet from week 6, but the trend was only significant from week 9 (P = 0·05–P< 0·07) with a significant difference in week 15 (P = 0·03). Energy intake and plasma concentrations of leptin, adiponectin and insulin did not differ between the dietary treatments (Table 5).

Fig. 2

Changes in (a) linoleic acid (LA), (b) arachidonic acid (AA) in liver phospholipids (PL), (c) liver 2-arachidonoylglycerol (AG), cerebral (d) 2-AG and (e) anandamide (AEA), and (f) energy intake, (g) feed efficiency, (h) weight gain, (i) final body weight …

Table 4

n-6 and n-3 profile of erythrocyte and liver phospholipids, and neutral lipids of epidldymal white adipose tissue (eWAT) in mice fed fish oil (FO) and soyabean oil (SO) salmon

Table 5

Physical and biochemical parameters in mice fed fish oil (FO) or soyabean oil (SO) salmon^†‡

Replacing FO with SO in feed for Atlantic salmon significantly lowered EPA and DHA in mice liver-PL, erythrocyte-PL and total lipids of eWAT, thereby decreasing the omega-3 index from 23 to 16 and increasing n-6 highly unsaturated fatty acids from 19 to 39 % (Table 4).

Adipose tissue accumulation and the adiposity index did not differ between mice fed the SO and FO salmon diets (Table 5). Staining eWAT and iWAT for the macrophage marker F4/80 showed considerably more F4/80-positive macrophages forming crown-like structures around adipocytes in mice fed the SO salmon diet than mice fed the FO salmon diet (Fig. 3). The SO salmon diet containing 8 en% LA resulted in larger adipocyte size in iWAT, but not in eWAT, than the FO salmon diet containing 1 en% LA (Fig. 3).

Fig. 3

Histology of epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) in mice fed the fish oil (FO) and soyabean oil (SO) salmon diets. (a) Immunostaining with the macrophage marker F4/80 in eWAT and iWAT of FO- and SO-fed mice. …

Discussion

Human consumption of soyabean oil has increased from 2·2 to 7·3 en% at the same time as the intake of EPA and DHA declined in the USA during the twentieth century⁽³⁾. Increasing intake of LA has been linked to obesity in both humans^{(10, 19, 21, 51)}and rodents^{(7, 10, 18, 19, 23, 52 – 54)}. In the present study, we found that mice fed the SO salmon diet with a high level of LA (8 en%) had higher liver concentrations of AA-PL and 2-AG. The elevated endocannabinoid activity in mice fed the SO salmon diet was associated with increased feed efficiency, higher weight gain and body weight (P < 0·8) compared with mice fed the FO salmon diet. We have previously shown that elevating dietary LA from 1 to 8 en% increases endocannabinoid production and induces obesity in mice fed high-fat diets, an effect that was reduced by adding 1 en% EPA and DHA to 8 en% LA diets⁽¹⁰⁾. The present and our previous findings are consistent with several reports that show how dietary fat alters endocannabinoid levels^{(5, 6, 11 – 14, 20, 55)}.

The findings of lower weight gain and body weight in mice fed the FO salmon diet compared with mice fed the SO salmon diet are in line with the general notion that fish oil rich in EPA and DHA limits high-fat diet-induced obesity in rodents^(26, 27), an effect that is associated with reduced tissue levels of AA-PL⁽²²⁾. The diet based on SO-enriched salmon resulted in larger adipocyte size in iWAT and more macrophage infiltration in eWAT than the FO-fed salmon diet. Diets enriched with n-3 PUFA have been demonstrated to reduce adipose tissue inflammation in diet-induced obesity^{(56 – 58)}. The mechanism by which EPA and DHA reduce macrophage-induced adipose tissue inflammation has recently been demonstrated to be mediated by the stimulation of the fatty acid receptor GPR120⁽⁵⁹⁾. EPA and DHA can be converted to metabolic products such as resolvins and protectins with anti-inflammatory actions independent of the state of obesity⁽⁶⁰⁾. Although both salmon diets contained relatively high amounts of EPA and DHA, the present data suggest that dietary LA of 8 en% decrease the anti-inflammatory properties of EPA and DHA. In keeping with the recent finding that sucrose counteracts the anti-inflammatory effect of fish oil in adipose tissue⁽⁶¹⁾, these data demonstrate that the background diet influences the anti-inflammatory properties of EPA and DHA.

Traditionally, farmed Atlantic salmon have consumed diets high in EPA and DHA and low in LA and α-linolenic acid. Introducing vegetable oils in farmed fish feed has altered the dietary fatty acid profile and fillet fatty acid composition⁽⁴⁵⁾. Consistent with prior reports^{(44 – 46)}, replacing fish oil with soyabean oil in feed for Atlantic salmon increased dietary LA from 1·5 to 21 en% in feed, and altered the fatty acid profile of salmon fillet and liver by increasing LA and decreasing EPA and DHA after 6 months of feeding. To our knowledge, we are the first to show that dietary LA from soyabean oil elevated 2-AG in the salmon liver. Consistent with previous reports replacing fish oil with soyabean oil⁽⁶²⁾ or a blend of vegetable oils^{(63 – 65)}, we found that Atlantic salmon fed soyabean oil accumulated significantly more TAG and total lipids in the liver than salmon fed fish oil. Liver is the main source of de novo fatty acid synthesis, and the activation of liver cannabinoid receptor 1 stimulates de novo fatty acid synthesis in mice by increasing the activity of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c), and its target enzymes fatty acid synthase and acetyl-CoA carboxylase⁽⁸⁾. Recent studies have demonstrated that replacing fish oil with vegetable oil in Atlantic salmon up-regulated the expression of SREBP-1c and fatty acid synthase in the salmon liver⁽⁶⁶⁾, affecting pathways of cholesterol and lipoprotein metabolism⁽⁶⁵⁾. It is therefore likely that an elevated endocannabinoid tone in the liver of salmon fed soyabean oil may have stimulated the hepatic de novo synthesis of fatty acids. In Atlantic salmon, replacing fishmeal and fish oil with high levels of plant protein (80 %) and a vegetable oil blend (70 %) of rapeseed, palm and linseed oil increased whole-body lipids, the visceral somatic index and liver lipid stores after 12 months of feeding⁽⁶³⁾ concomitant with a significant lower body weight⁽⁶⁷⁾. In another study, replacing fish oil with a vegetable oil blend (rapeseed, palm and linseed oil) increased body weight in Atlantic salmon receiving vegetable oil compared with fish oil possibly due to the long-term effects increasing nutrient utilisation⁽⁴⁴⁾. We did not find any effect on body weight or the visceral somatic index in salmon when soyabean oil replaced fish oil for 6 months, indicating interacting effects between oil source and protein source, or length of feeding required to affect body weights or visceral fat stores in Atlantic salmon^(44, 63).

There have been increasing concerns about the decreasing content of EPA and DHA in farmed Atlantic salmon. Norwegian surveillance data⁽⁶⁸⁾report a moderate increase in LA levels in the fillets of farmed Atlantic salmon from 1·1 g/100 g in 2005 to 1·6 g/100 g in 2010 and a decrease in EPA+DHA from 2·7 g/100 g to 2·1 g/100 g in the same period. In the present study, Atlantic salmon was fed soyabean oil, a major dietary source of LA, resulting in 2·5 g LA and 0·7 g EPA+DHA/100 g fillet, representing an extreme model where vegetable oils replace fish oil in feed for Atlantic salmon. Although recognised as suitable alternatives to fish oil in feed for Atlantic salmon, the present findings of elevated liver endocannabinoid and lipid accumulation in salmon fed soyabean oil suggest that future fish oil replacement in farmed Atlantic salmon should pay attention to the choice of vegetable oils with regard to LA content. However, although lower than the FO salmon diet, mice fed the SO salmon diet for 16 weeks had a high omega-3 index and low n-6 highly unsaturated fatty acids (16 and 39 %, respectively), indicating that farmed Atlantic salmon fed vegetable oil remain a significant dietary source of EPA and DHA.

In summary, replacing fish oil with soyabean oil in feed for Atlantic salmon introduces high dietary levels of LA in Atlantic salmon and elevates AA, endocannabinoid activity and TAG accumulation in the salmon liver. Mice consuming Atlantic salmon fed soyabean oil have higher liver levels of AA-PL and 2-AG, higher feed efficiency, higher weight gain and more adipose tissue inflammation than mice fed the FO salmon diet. Thus, lower dietary levels of LA may improve metabolic functions associated with obesity.