Alterations of EPA content in brain after HFD diet

The most important and at the same time surprising result of this study is significant reduction of EPA in serum, brain and other tissues in HFD compared to SD mice. Simultaneously, every other PUFA were increased or not changed in serum and brain of the HFD mice, possibly due to their higher concentration in high-fat chow (Table 2). Jansen et al. [30] suggested that mice with obesity have a higher needs for essential fatty acids than non-obese animals. EPA and DHA are the subjects of enzymatic oxidation, which results in the formation of eicosanoids and docosanoids, named oxylipins, which have anti-inflammatory properties. EPA is a precursor of three series of eicosanoids and related peroxy-fatty acids, a molecules with most powerful anti-inflammatory properties [6, 25]. In turn, ARA derived eicosanoids displays a pro-inflammatory properties [29, 31]. Thus, decreased EPA in brain may result in increase of inflammation in this organ. Another, important function of long chain PUFA is impact on membrane flexibility, fluidity and permeability as well as, assure the passive transport by the membrane [6, 25, 32]. They are main compounds of brain membranes. DHA and ARA together are almost one fifth of the brain dry weight [6, 29]. The composition of membrane lipids appears to influence the mental health, what relies on lipid-protein interactions within the membrane. It may influence neurotransmitter release and reuptake, and membrane receptors function [33]. Supplementation of EPA enhances proliferation in neural stem cells [25]. However, when EPA enters into the brain it is rapidly oxidized [34, 35], in contrast to DHA [36]. This is not the case with DHA and ARA. Infusion of 14C-EPA in situ showed that EPA has a half-life only 5 days in brain phospholipids, while DHA and ARA have 33 and 42 days, respectively [37]. Therefore, the research on ADHD, depression and brain trauma, suggests that EPA supplementation should be even higher than DHA to maintain its appropriate concentration in blood, and thus accessibility for the brain [38].

Alterations of particular lipid fraction in mice brain after HFD diet

In this study for the first time the profile of fatty acids in particular lipid fractions in brain of mice treated by HFD was investigated. One of the used chemical procedure of lipid fraction separation was Kaluzny et al. [27] SPE method that allowed to obtain three lipid fractions: AG, PL and FFA. Participation of particular lipid fractions in HFD mice were changed in comparison to SD mice brain (Fig. 3). In HFD mice we found statistically significant increase of AG fraction (Fig. 3), that is mainly constituted by diacylglycerols (DAG) and triacylglycerols (TAG) [39]. The elevated levels of AG can be attributed to higher level of MUFA in HFD mice (Table 3). Also, the increased levels of acylglycerols were observed by Borg et al. [39], in the hypothalamus of HFD mice comparing to animals fed low fat diet. DAG is a product of metabolism of both phospholipids and TAG, and it is implicated in the development of central insulin resistance in the brain [39].

PL are about 70% of lipids separated by Kaluzny et al. [27] SPE method, but there was no significant difference between SD and HFD mice (Fig. 3). PL are extensively synthetized in the brain [40], constituting the majority of structural lipids located in the phospholipid bilayer [29]. The level of EPA in PL fraction in HFD brain was almost three time lower compared to SD mice brain (Table 2). By contrast we observed significant increase of EPA in HFD mice brain among FFA fraction (Table 3). Perhaps, free EPA originate from hydrolysis of PL and SPL, where its content is decreased. Interestingly, despite EPA and DHA belong to long chain n-3 PUFA, their participation in specific/particular lipid fraction is very different. DHA is most abundant in polar lipids, which are building cellular membranes of brain [25], in order to facilitate transport through the mitochondria membrane and decrease the levels of mitochondrial reactive oxygen species [41]. DHA is about 40% of all PUFAs in the brain [42, 43], what was also observed in our study (Table 2). DHA due to additional double bond in its structure, and longer aliphatic acid than EPA, takes up a lot more space in membrane, which results in the greater fluidity of brain membranes. This allows for more effective receptor functioning and signal transmission into the interior of the nerve cells [44].

Second method of separation of brain lipids used in our study was modification of Bodennec et al. [28] method. In this way we received five fractions, including neutral lipids, Cer, normal and α-hydroxy free fatty acids, GSPL and sphingomyelin. In our study we focused on SPL, due to their important functions in the brain [29]. Sphingolipids is a wide lipid group including Cer, SM and GSPL [29]. SM and GSPL are mainly localized in the plasma membrane, acting as determinants of membrane permeability and fluidity [45]. Concentration of sphingolipid in brain is very high due to their function in creation the lipid rafts [29]. GSPL in their structure have various carbohydrates attached to the ceramide [29], and one third of the GSPL are gangliosides, which in neuronal membranes constitute 12% of total lipid content [46]. Their function in membrane is protein modulation, synaptic transmission, cell-cell adhesion, neural development and differentiation, axonal growth, and receptor regulation [47]. Perhaps, that is why they were in the highest levels among examined sphingolipids in the brains of examined mice (Fig. 4). GSPL levels in HFD mice were significantly decreased that may lead to alterations of the above mentioned important function in the brain In turn, elevated levels of Cer in HFD brain compared to SD brain (Fig. 4) is associated with lipotoxicity of Cer including induction of apoptosis, inflammation and central insulin resistance in the brain [39].

Dominant PUFA among SM fraction was DHA, which plays a significant role in cells signaling as a component of lipid rafts [48]. Also, due to DHA greater flexibility it is more readily incorporated into the glycosphingolipids membrane [49]. In GSPL and Cer fraction the levels of EPA and DHA were significantly lower than ARA level (Table 3). In contrast to DHA and ARA, in each determined sphingolipid fraction we observed significant decrease of the levels of EPA in brain of HFD mice (Table 3). One reason for decreased EPA content in brain of mice after HFD may be its lower availability in blood. Moreover, the other reasons of lower EPA levels can be the involvement of EPA in lipid metabolism in the brain, including β-oxidation, elongation/desaturation to docosapentaenoic acid (22:5n-3; DPA), which is precursor of DHA [50]. EPA is such an important acid not only due to the above-mentioned properties, but also affects the functioning of the brain. Studies on rat brains showed that EPA is increased in cortical tissues, improved spatial memory in the aged rats and restores log-term potentiation [51]. The above-mentioned data suggest that decreased levels of EPA in PL, and sphingolipids in brain of HFD mice may contribute to brain dysfunction.

Changes of PUFA/EPA in other mice organs

Consumption of food reach in fat leads to fat gain and increased body weight, especially diets containing more than 30% of total energy as fat lead to the development of obesity [52]. However, it has been reported that not every fat is obesogenic and the fatty acid profile rather than the energy from fat is crucial in the development of obesity [52]. On the other hand, some studies did not show differences between body-weight gain of the animals consuming food containing various fatty acids [52].

Some authors described elevated levels of TAG and FFA in serum mice after high-fat diet [53, 54]. TAG and FFA are responsible for induction of oxidative stress, lipotoxicity, dyslipidemia, insulin resistance and diabetes [55]. Also, our study showed significantly increased serum total fatty acids, that are possibly included both in TAG as well as FFA fractions. The excessive deposition of lipids in cell other than adipocytes leads to cellular stress, dysfunction, and sometimes to apoptotic cell death termed lipotoxicity. This process is involved in the development of many diseases [39]. The fatty acid composition of various lipids is often reflective of the fatty acid composition in consumed food [56]. However, despite that the concentration of total FA in high fat chow was four time higher than in standard chow (Table 2), the levels of EPA were significantly decreased in serum and all analyzed HFD mice organs (Tables 2, 3).

Significant differences in EPA content in liver and three adipose tissues depots (Table 2) may also lead to increase of adiponectin secretion in adipose area [57], that could increase the risk of comorbidities of obesity, including cardiovascular disease and insulin resistance [58]. What is more, adequate amount of EPA in intake diet prevents obesity by inducing mitochondrial biogenesis and beta-oxidation in adipocytes [59].