Cyclooxygenase-2 and n-6 PUFA are lower and DHA is higher in the cortex of fat-1 mice

https://doi.org/10.1016/j.neuint.2009.12.022Get rights and content

Abstract

Neuroinflammation is believed to play an important role in neurological diseases such as Alzheimer's disease (AD). Growing evidence suggests that n-3 PUFA have protective effects by inhibiting inflammatory processes including synthesis of eicosanoids from arachidonic acid. There is also some evidence suggesting that inflammatory mediators associated with the arachidonic acid cascade may be modulated by n-3 PUFA in healthy animals. Therefore, in this study, the effect of n-3 PUFA on brain cortex fatty acid composition, and on the expression of calcium-dependant cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) was assessed in the transgenic fat-1 mouse. Phospholipid fatty acid composition was determined by thin layer and gas chromatography, while cortical cPLA2 and COX-2 were determined by Western blotting. 22:6 n-3 levels were up to 220% higher while n-6 PUFA levels were up to 77% lower in fat-1 phospholipid fractions of the cortex as compared to wildtype (WT) mice. COX-2 protein levels were 25% lower in fat-1 as compared to WT mice (p = 0.02), but cPLA2 expression levels did not change. Our results suggest that this model could be used to investigate mechanisms by which n-3 PUFA regulate neuroinflammation.

Introduction

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are polyunsaturated fatty acids (PUFA) which are highly enriched in brain plasma membrane phospholipids (Diau et al., 2005). DHA and AA are 22 and 20 carbon PUFA, respectively. DHA belongs to the n-3 while AA belongs to the n-6 family of fatty acids. The n-3 and n-6 nomenclature refers to the position of the first double bond relative to the methyl terminus. Relative to the diet, DHA is primarily obtained from marine oils while AA is found in animal fat.

It has been reported that DHA may be protective against neurological diseases and cognitive decline (Lim et al., 2005a, van Gelder et al., 2007). In contrast, AA is associated with inflammatory events in the brain which may increase risk for neurodegenerative diseases (Farooqui et al., 2007). After being cleaved and released from the plasma membrane by phospholipase A2 (PLA2), AA can be metabolized into pro-inflammatory prostaglandins by cyclooxygenase-2 (COX-2) (Farooqui et al., 2007). Deprivation of dietary n-3 PUFA in rats for 15 weeks, resulting in lower brain n-3 PUFA and higher n-6 PUFA, has been shown to increase the expression of enzymes associated with the AA inflammatory pathway such as cytosolic phospholipase A2 (cPLA2) and COX-2 (Rao et al., 2007).

It is important to note that n-3 PUFA and n-6 PUFA cannot be interconverted or formed de novo by mammals due to the lack of appropriate desaturase enzymes. Linoleic and α-linolenic acid obtained from the diet can be elongated and desaturated to form long chain PUFA including, AA and DHA but the rate of conversion is very low (Pawlosky et al., 2001, James et al., 1993).

The fat-1 transgenic mouse is able to convert n-6 to n-3 PUFA due to the expression of the fat-1 gene from Caenorhabditis elegans which encodes for an n-3 desaturase (Kang et al., 2004). n-3 PUFA enrichment and n-6 PUFA reduction have been reported in total lipid extracts from whole brains of the fat-1 mouse which appear to mimic effects observed after n-3 feeding (Kang et al., 2004, Taha et al., 2008, Lim et al., 2005b). These observations suggest a potential utility of the fat-1 mouse model to study the role of DHA and AA in brain physiology and disease. Given that these major n-3 and n-6 fatty acids play a role in initiating anti-inflammatory and pro-inflammatory signalling pathways, detailed phenotypic characterization of the origins of these biologically active fatty acids is needed, which reside in membrane phospholipids is lacking. Furthermore, no fatty acid data has been reported in the literature for specific brain regions in the fat-1 mouse. These data are fundamental to validating the utility of the fat-1 genetic model to study the relationship between AA, DHA and inflammatory processes. Therefore, the objectives of this study are to determine the phospholipid fatty acid composition in the brain cortex and effects of the fat-1 gene on cPLA2 and COX-2 expression in the cortex.

Section snippets

Animals and diet

Heterozygous fat-1 males on a mixed C57BL/6 × C3H background were bred with wildtype (WT) C57BL/6 females (Charles River, Saint-Constant, Quebec, Canada) to produce heterozygous fat-1+/− and WT mice. All mice were fed a modified n-6 PUFA enriched and n-3 PUFA deficient AIN-93G diet containing 10% of safflower oil instead of 7% soybean oil (Product #D04092701; Research Diets, New Brunswick, NJ). This model permits the characterization of effects of n-3 deficiency in WT mice while fat-1 mice are

Fatty acid composition of phospholipids and sphingomyelin in the cortex of the fat-1 mouse

As shown in Fig. 1, DHA was approximately 3 times higher in phospholipid and sphingomyelin fractions of the fat-1 mouse cortex when compared to WT (p < 0.05). Because the fat-1 mouse converts n-6 PUFA to n-3 PUFA, this also results in 26%, 49% and 77% lower (p < 0.05) levels of n-6 PUFA in glycerophosphocholine, glycerophosphoethanolamine and glycerophosphoserine, respectively, in fat-1 mice. The lower DHA in WT mice was compensated by the presence of the 22 carbon, 5 double bond, n-6 PUFA,

Discussion

In this study, detailed fatty acid analysis of the cortex showed higher levels of DHA in fat-1 mice compared to n-3 PUFA deficient WT controls. Although total lipid analyses would have been simpler, it does not provide the level of detail in order to ascertain whether changes in fatty acid composition due the fat-1 gene are comparable to existing literature values. Fatty acids results demonstrate that n-3 PUFA, specifically DHA, are higher in all phospholipids fractions of the fat-1 mouse

Acknowledgments

Funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants were provided to D.W.L. Ma and R.P. Bazinet. Funding from the J.P. Bickel Foundation for Medical Research and Connaught Fund were also provided to D.W.L. Ma. C. Boudrault is supported by a Canada Graduate Scholarship through NSERC.

References (40)

Cited by (22)

  • n-3 long-chain PUFA-containing phospholipids and neuroprotection

    2019, Omega Fatty Acids in Brain and Neurological Health
  • Interplay between lipid mediators and immune system in the promotion of brain self-repair

    2017, Role of the Mediterranean Diet in the Brain and Neurodegenerative Diseases
  • Modulation of brain PUFA content in different experimental models of mice

    2016, Prostaglandins Leukotrienes and Essential Fatty Acids
    Citation Excerpt :

    The fatty acid composition varied within the different brain regions confirming several previous reports [5,8,31]. In response to dietary n-3 PUFA deficiency or to a genetic-driven enrichment in n-3 PUFAs, all brain regions were impacted in terms of AA and DHA variations as already reported [5,6,12,32–37]. Yet, some structures were more affected than others.

  • Could heterotopic ossification be prevented by varying dietary n-3/n-6 polyunsaturated fatty acid ratio: A novel perspective to its treatment?

    2013, Medical Hypotheses
    Citation Excerpt :

    Combined with the previously described notion that PGE2, derived from arachidonic acid, is believed to mediate the inflammation in the HO formation as the key factor, Lowering the dietary (n-6)/(n-3) PUFAs ratio may reduce arachidonic acid synthesis, subsequently impair its conversion to PGE2 by COX-2, which may prevent HO. In addition, recent investigations from in vitro culture system and transgenic animal models proposed that modifying the dietary (n-6)/(n-3) PUFA ratio to a lower level may block COX-2 expression and its activity [33–36]. A clinical trial has been reported by Caughey et al. that enriching diet with n-3 PUFAs by fish oil supplement, increases the cyclooxygenase inhibitory activity of paracetamol in rheumatoid arthritis patients [37], whilst Horia et al. suggested that n-3 PUFAs could exert a complementary inhibitory on COX-2 activity in breast cancer cell line [38].

View all citing articles on Scopus
View full text