Mantenimiento preventivo

The composition of fats within the diet has previously been scrutinised for their health benefits (Calder, 2006; Ruxton et al., 2007; Khorsan et al., 2014; Elagizi et al., 2018). Populations with a diet rich in ω-3 PUFAs EPA and DHA have been shown to have a lower incidence of chronic non-communicable diseases (Simopoulos, 1999). The Mediterranean Diet for example, high in PUFAs and MUFAs, has been demonstrated to have beneficial effects on individuals with cardiovascular disease (CVD) by reducing the associated risk factors in comparison to the levels associated with a low- fat diet (Estruch et al., 2006).

Considerable research has been undertaken in an attempt to establish whether it is the dietary ω-6 LA:ω-3 ALA ratio that has an impact upon disease manifestation in humans. Simopoulos (2002) concluded that a lower ratio reduced the risk of many chronic diseases. However, a cross-sectional study completed as part of the Tehran lipid and glucose study in Iran concluded that there was no association between the dietary ω-6/ω-3 PUFA ratio and metabolic syndrome (MetS; Mirmiran et al., 2012). Other researchers have found that intake of ω-6 PUFAs did not influence the beneficial effect of increased ω-3 PUFAs intake on the risk of CVD and chronic heart disease (CHD; Mozaffarian et al., 2005; Fontes et al., 2015). Mirmiran et al. (2012) found an inverse association between both ω-3 PUFA ALA and ω-6 PUFAs with MetS, the highest consumptions having lowest prevalence. In this study, no significant associations were found however between ω-3 EPA or ω-3 DHA and MetS. However, dietary intake of the ω-6 and ω-3 PUFAs was assessed using a food-frequency questionnaire (FFQ). This approach to data collection is inherently flawed because they rely on subject recall and it is difficult to achieve quantifiably precise records and they are not suitable when seeking to establish epigenetic relationships in detail. Evidence that ω-3 PUFAs, such as EPA and DHA, can modulate inflammation has come from in vitro work (Endres et al., 1989; Trebble et al., 2003; Verlengia et al., 2004), animal models (Yaqoob and Calder, 1995; Al-Khalifa et al., 2012; Olson et al., 2013; Richard et al., 2016) and human studies (Endres et al., 1989; Tartibian, Maleki and Abbasi, 2011; Itariu et al., 2012); although the latter evidence is weaker as a

consequence of more complex environmental factors. The mechanisms through which ω-3 PUFAs promote an anti-inflammatory environment within the body are multifaceted and complex (Calder, 2015), and are illustrated in Figure 2-5.

Inflammatory diseases are often characterised with chronic activation of NFkB pathway (Lawrence, 2009). In its inactive form, NFkB is stored in the cytoplasm covalently bonded to its inhibitory subunit IkB (Jacobs and Harrison, 1998). After activation, IkB is phosphorylated by IKKs and this allows for the translocation of NFkB to the nucleus (Israel, 2010). ω-3 PUFAs have been shown reduce activation of NFkB by multiple mechanisms. Ligand bound PPARy can interact with NFkB by preventing its translocation to the nucleus (Calder, 2012b). EPA has also been shown to inhibit the translocation of NFkB subunits to the nucleus directly. Zhao et al., (2004) showed THP-1 cells treated with EPA had decreased translocation of the p65 subunit to the nucleus following LPS stimulation; pre-stimulation with EPA did not alter cytoplasm p65 and p50 content. EPA treatment resulted in lower NFkB activity after LPS stimulation and reduced TNFα mRNA expression and cytokine production (Zhao et al., 2004). EPA treatment also supressed the phosphorylation of IkB-a in the cytoplasm, indicating that degradation of IkB-a and translocation of NFkB to the nucleus may be prevented by EPA through decreased phosphorylation of IkBa. Reduced activation of NFkB by ω-3 PUFAs in response to inflammation ultimately results in reduced expression of its target genes, such as cytokines. Treatment in vivo with EPA and DHA can modulate the expression of multiple inflammatory genes within the NFkB pathway (Allam-Ndoul et al., 2016). In vitro models have demonstrated that treatment with EPA and/or DHA reduces cytokine production after stimulation with LPS.

Treatment of cells ex vivo with an inflammatory stimulus, for example LPS, and measurement cytokine production, can be used to measure the effect of in vivo supplementation with ω-3 PUFAs on the inflammatory response. Decreased production of TNFa, TNFR2, IL6, IL-1b, IL2, IFNg and CRP have been observed when cells are stimulated ex vivo after ω-3 PUFA supplementation (Verlengia et al., 2004). Some studies have examined the effect of EPA and DHA separately, DHA is shown to have a greater inhibitory effect on TNFa, IL-1b IL6, (Weldon et al., 2007). Gene expression for TNFa, IL1b and IL6 were measured and were consistent with the

cytokine data (Weldon et al., 2007). Whereas, EPA decreases production of IL2 and IFNg to a greater extent than DHA (Verlengia et al., 2004). The dosage of supplementation has also been shown to have an effect; three doses of ω-3 PUFAs over 12 weeks in males resulted in a 'U-shaped' dose-response curve with maximum inhibitory effects TNFα and IL6 production at 1g/day compared to the 0.3 and 2g/day (Trebble et al., 2003).

Modulation of inflammatory gene expression also occurs through transcription factors. Once the ω-3 PUFAs are incorporated in the cell membrane they can be released into the cell and can interact with these transcription factors, such as PPAR, in the regulation of gene expression. Activation of PPAR occurs when ligands, including ω- 3 PUFAs and eicosanoids, are non-covalently bonded to them. PPARg and PPARa are the most understood of the PPAR isoforms (Tyagi et al., 2011). Of interest here, PPARg is expressed in inflammatory cells and has an anti-inflammatory action. In a cancer cell line, treatment with EPA and DHA have shown to increase expression of PPARg (Rovito et al., 2013). DHA interacts with PPARg resulting in reduced cytokine production after stimulation (Li et al., 2005). PPARg is also involved in regulating metabolism in adipocytes with downstream effects promoting insulin sensitivity and blood triglyceride concentrations (Poynter and Daynes, 1998; Dunning et al., 2014; Chiazza and Collino, 2016). Expression of PPARg is modulated by DNA methylation within its promotor (Fujiki et al., 2009). In a case-control study of T2DM patients, DNA methylation was significantly different in genes involved in inflammation, among other pathways; differential methylation was also shown in T2DM candidate genes, including PPARG (Nilsson et al., 2014).

Alterations to the fluidity of the cell membrane, through changes in fatty acid composition, has an impact on the function of proteins within the membrane, cell signalling and gene expression. Specific genes have been found to be differentially expressed by alterations in ω-3 PUFA levels. The membrane glycoprotein which promotes inflammation in monocytes and macrophages, CD36 “cluster of differentiation 36”, has been found to be differentially expressed as the result of ω-3 PUFA supplementation. CD36 is a membrane glycoprotein expressed on the surface of many cells that are active in fatty acid metabolism, such as adipocytes, muscle cells,

functions include the uptake of long-chain PUFAs, regulation of angiogenesis, scavenger in innate immunity, and has a key role in foam cell formation (Febbraio, Hajjar and Silverstein, 2001). In human skeletal muscle and macrophages, high levels of CD36 have been correlated with insulin resistance (Bonen et al., 2004; Liang et al., 2004). However, this correlation may be due to the up-regulation of CD36 that occurs in response to high fat feeding (Greenwalt, Scheck and Rhinehart-Jones, 1995) and diabetes (Greenwalt, Scheck and Rhinehart-Jones, 1995; Van Nieuwenhoven, Van der Vusse and Glatz, 1996) as a consequence of the increase fatty acid utilisation, demonstrated in cell culture with long-chain fatty acid treatment (Sfeir et al., 1997). In a rat model of MetS using spontaneously hypertensive rats (SH-rats), in which expression levels of CD36 are undetectable in adipocyte plasma membranes, a diet rich in ω-3 PUFAs was shown to increase CD36 mRNA expression to comparable levels to those of Kyoto-Wistar rats (KW-rats) on a control diet (Alexander Aguilera et al., 2006). Metabolic parameters of the SH-rats were increased compared to the KW- rats prior to diet changes; they had increased blood pressure, serum insulin, FFAs and triglyceride levels. After the diet intervention, the SH-rats fed ω-3 PUFAs had significantly lower serum insulin, FFAs, triglycerides, total cholesterol, HDL, LDL, and total lipids than the SH-rats on a canola-corn control diet, giving a metabolic profile similar to the KW-rats. Serum glucose measurements were not found to be different across the three groups. This study is an example of one gene which has altered expression levels following the addition of ω-3 PUFA to the diet.

Agreement with these results has been shown in cell culture, increased CD36 mRNA expression as the result of separate EPA and DHA treatment has also been shown in THP-1 macrophages, in a dose-dependent manner (Vallvé et al., 2002). However, when expression of CD36 was measured by assessing mRNA levels in PBMCs isolated from healthy human males and females who’s diet had been supplemented with combined EPA (1.8g/day) and DHA (0.4g/day) for 26 weeks; a decrease in CD36 mRNA expression was observed (Bouwens et al., 2009). Using a micro-array, the expression of 900 genes uniquely changed as a result of the ω-3 PUFA supplementation compared to a placebo. A decrease in expression of genes involved in inflammatory pathways was observed. However, no changes in plasma C-Reactive protein (CPR), a measure of inflammation, were seen in this cohort and no other

measures of inflammation were investigated. The study also found a decrease in the expression of PDK4, LTA4H, ADFP, CD14 and HIF1a (Bouwens et al., 2009).

Another cell membrane bound protein important in the anti-inflammatory effects of ω- 3 PUFAs is G-Protein-Coupled receptor 120 (GPR120). GPR120 functions as a receptor for unsaturated long chain free fatty acids. EPA and DHA have been found to exert their potent anti-inflammatory effects through GPR120 (Oh et al., 2010). It was shown that GPR120 knockout mice did not show inhibition of inflammation from ω-3 supplementation, whereas the wild-type mice had reduced inflammation and enhanced insulin sensitivity as a result of the supplementation. Two non-synonymous variants of GPR120 have been found to be associated with diabetes; Ichimura et al. (2012) also found that in GPR120 knockout mice fed on a high fat diet showed increased body weight compared to the wild-type mice fed on the same diet.

Evidence suggests that one of the ways in which ω-3 PUFAs exert these effects is through changes in gene expression as a result of varied transcription factor activation (Bouwens et al., 2009; Rudkowska et al., 2013), however, they may also be a consequence of changes in epigenetic mechanisms, such as DNA methylation.

Figure 2-5 Mechanisms Omega 3 polyunsaturated fatty acids (ω-3 PUFAs) action to promote an anti-inflammatory environment. G-coupled protein receptor 120 (GPR120); nuclear factor kappa B (NFkB); omega 6 polyunsaturated fatty acids (ω-6 PUFAs); Specialised pro-resolving mediators (SPMs); toll like receptor 4 (TRL4).)