We have previously shown that modeling the increase in human consumption of dietary LNA from 1 to 8 en% LNA during the last century [23] caused excessive endocannabinoid levels and also induces obesity in mice fed diets of 35 and 60 en% fat [14]. To our knowledge, we demonstrate here for the first time that an LFD can be made obesogenic by inclusion of 8 en% LNA, subsequently stimulating excessive endocannabinoid activity in the liver and weight gain. In the present study we report that isocalorically increasing dietary LNA from 1 to 8 en% elevated tissue levels ARA–PL, liver 2-AG and AEA regardless of changes in total dietary fat, and increased feed efficiency, caused higher weight gain and elevated plasma leptin in mice fed the LFD. We also observed a tendency of increased cell size and macrophage infiltration in eWAT in mice fed 8 en% LNA.

The percent of n-6 in HUFA can be used as an indicator of disease risk as it models the relative amounts of tissue ARA–PL available as precursors for eicosanoid derivatives of ARA [28]. We used the Lands equation [25] to predict tissue ARA–PL composition as the % n-6 HUFA from dietary intakes of n-6 and n-3 PUFA to model availability of endocannabinoid precursors with good concurrence between calculated and experimental values. Here we modeled the increase in US dietary intakes of LNA from 1 to 8 en% [23] in our animal diets, and showed in mice, that elevating dietary LNA to 8 en% increased ARA–PL in liver, RBC and adipose tissue to levels currently found in Americans [29], and induced elevations in liver 2-AG and AEA. Elevating endocannabinoid levels by dietary LNA to alter the availability of ARA in the phospholipid precursor pool is consistent with several studies where dietary fatty acids alter endocannabinoid levels [30–36], and our previous work where we selectively altered LNA and raised dietary omega-3 HUFA both in mice and salmon [14, 15]. We did not observe any differences in food intake or endocannabinoid levels in the cerebral cortex, suggesting that the effects of increased endocannabinoid levels were caused by effects in peripheral tissue. The adult brain is less susceptible to dietary manipulations than the young brain [37, 38]. The lower sensitivity of dietary LNA on brain endocannabinoid levels compared to peripheral tissues confirm that brain lipids levels are less influenced by changes in dietary fatty acids and that this may be reflected also in the endocannabinoids [39, 40].

Dietary LNA of 8 en%, independent of dietary fat content, significantly elevated plasma leptin levels compared to 1 en% LNA. Plasma adiponectin levels were less affected by dietary LNA, suggesting that leptin is a more sensitive marker of early obesity development. Mice fed an LFD had similar plasma concentrations of adiponectin per gram adipose tissue despite higher WAT and adiposity in mice fed 8 en% LNA compared to 1 en% LNA. This result suggests that mice fed a high LNA content secrete less adiponectin per gram fat, in line with the obesity-prone phenotype of the 8 en% LNA diet.

We find that the LFD containing 8 en% LNA significantly increased feed efficiency compared to the 1 en% LNA diet causing the animals to gain more weight per calorie consumed. The increased feed efficiency and weight gain in mice fed 8 en% LNA in the LFD resulted in a similar body weight as mice fed 35 en% fat, significantly higher than mice fed 1 en% LNA in an LFD. Dietary LNA of 1 en% has been found to reverse the obesogenic properties of a high fat diet [14]. In line with previous studies, our findings in the LFD support the notion that weight gain involves mechanisms that depend more on the composition of dietary fat than the total amount of fat in the diet [12, 30, 41–43]. Increased intake of LNA has been linked to increased prevalence of obesity [26, 41] and the primary dietary sources of LNA; soybean oil, poultry and shortening were positively correlated to increasing rates of obesity occurring in the US during the 20th century [14]. Replacing saturated fats with LNA-rich vegetable oil increased body weight in veterans [44] and ARA levels in adipose tissue were positively associated with BMI in children [45]. In animals, vegetable oils rich in LNA (usually soybean oil; 55 % LNA, and safflower oil; 75 % LNA) elevated food intake [14, 15, 46], induced weight gain [14, 15, 30, 41, 42, 47] and increased lipogenic enzyme activity in the liver [10, 48]. Our findings imply that low fat diets could be made more effective in reducing adiposity if LNA were lowered to near 1 en%. Indeed, total dietary fat intake may not need to be lowered if LNA is selectively lowered.

In contrast to what we previously reported [14], 1 en% LNA in the MFD did not prevent weight gain and adiposity compared to MFD 8 en% LNA. In the present study animals were fed the experimental diets from 6 weeks of age, whereas in our previous study animals were exposed to the dietary treatments from the last week of pregnancy to 17 weeks of age [14]. D’Asti et al. [49] demonstrated how quantity and quality of maternal dietary fat during the perinatal period directly influences neonatal metabolism, fatty acid composition in phospholipids and sensitivity to endocannabinoid system manipulation. Massiera et al. [50] found a gradual transgenerational increase in adiposity in mice fed a “western-like” diet of 35 en% fat containing 18 en% LA for four generations. Thus the adipogenic effect of LNA appears to be higher when prenatally exposed [14]. Additionally, in the present study, mice were fed a standard NIH #31M diet based on soybean oil prior to arrival at our facility (Taconic, Denmark). Calculations by the Lands equation [25] show that the NIH #31M diet results in 72 % n-6 HUFA in tissue phospholipids, identical to our diets containing 8 en% LNA. Metabolic changes such as induced lipogenesis and deposition of lipid droplets in response to increased endocannabinoid activity have been shown to occur before onset of obesity [10, 12, 51]. It is possible that an already elevated endocannabinoid activity present in the animals upon arrival to our facility may have overridden the anti-adipogenic properties of the 1 en% LNA diet, when the MFD contained abundant carbohydrates [42]. The latter might be of importance as it has been demonstrated that the obesogenic potential of diets rich in both n-6 and n-3 PUFA is elevated by increasing the levels of dietary carbohydrates, and thereby raising the insulin/glucagon ratio translated into reduced cAMP signaling [42, 52]. cAMP signaling has been demonstrated to play a pivotal role in controlling the production of both pro- and anti-adipogenic prostaglandins both in vivo and in vitro [42]. Collectively these results illustrate the importance of the type of fat and carbohydrates in the background diet, and how these can influence the obesogenic properties of LNA. Mice fed an LFD of 8 en% LNA started to gain considerably more weight than mice fed 1 en% LNA after 8 weeks of feeding. Thus a longer feeding period may be necessary to wash out excessive tissue ARA caused by the NIH #31M diet, before a beneficial effect of a low LNA diet (1 en%) may occur. Although body weight did not differ, elevated liver 2-AG and AEA, larger adipocytes, higher macrophage accumulation in WAT and elevated plasma leptin levels in mice fed 8 en% may suggest a more obesity prone phenotype and a higher risk of developing obesity and metabolic complications associated with obesity compared to mice fed same amount of fat but with only 1 en% LNA.

One of the major limitations when working with dietary fat is to relate the observed differences to one fatty acid. There are no inert oils which can be added to a diet to target one single fatty acid to be different in a diet. We did extensive analyses of various fats and oils available on the market to establish the fatty acid profile of the products and carefully mixed several oils to isolate LNA as controlled variable via isocaloric replacement of saturated fat (in the medium fat diet). It was not possible to obtain similar content of MUFA in the low fat diets since the low fat diet contained 8 en% fat as LA of the total of 12.5 en% fat. Although our diets contained different amounts of SFA and MUFA, SFA were covariable in the LFD and MUFA were covariable in the MFD, thus strengthening our conclusion that LNA is the major dietary fatty acid affecting endocannabinoid levels and induces weight gain. If there were other fatty acids (MUFA or SFA) that strongly influenced endocannabinoid levels and adiposity we would expect the differences between the dietary treatments to be less distinct. Another limitation with our study is the absence of endocannabinoid measurements in adipose tissue, making it difficult to conclude if the observed elevation of endocannabinoid levels in the liver is a consequence rather than the cause of obesity. In order to establish a cause–effect relationship between a diet induced elevation in endocannabinoid levels and the observed obese phenotype in mice fed the LFD of 8 en% LNA a follow-up study should be carried out using a CB1 antagonist.

Reducing dietary LNA from 8 to 1 en% with a concomitant decrease in ALA from 1 to 0.3 en% significantly increased EPA–PL concentrations in RBC (three-fold) and liver, increasing the n-3 index from 5 to 8 and 9 in MFD and LFD respectively. We have previously reported that reducing LNA from 8 to 1 en% increased EPA and reduced ARA in liver and RBC similarly to supplementing an 8 en% LNA diet with 1 en% EPA + DHA [14]. Consistent with previous reports [53–58], our results indicate that the elongation and desaturation of ALA to EPA and DHA are considerably more effective when dietary LNA is 1 en%. Hence to improve tissue EPA and DHA concentrations, reduce ARA–PL and consequently decrease endocannabinoid production, emphasis should be on lowering dietary LNA in addition to dietary supplementation with EPA and DHA. In conclusion, a dietary approach by reducing substrate availability for endocannabinoid synthesis provides a safe and preventative alternative to decrease hyperactivity of the endocannabinoid system, and consequently decrease or prevent obesity.