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Mariniello, Katia (2011) Comparative study of synthesis and incorporation of omega-3 and -6 long chain polyunsaturated fatty acids by THP-1 and HT29 cells with a specific focus on the influence of retinoids. Doctoral thesis, London Metropolitan University.
In vitro cultured cells have been used as models in order to study cellular responses to omega-3 and -6 polyunsaturated fatty acids (PUFA). Some studies tested α-linolenic (ALA) and linoleic (LA) acids, others preformed eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA). Immune responses, cell proliferation, apoptosis and differentiation, as well as lipid metabolism and cell signalling have been investigated. Omega-3 and -6 PUFA have been shown to be involved in gene and protein expression, cellular transduction, cytokines and eicosanoid production. Nevertheless, most of the studies did not investigate fatty acid composition of cell membrane phospholipids. Hence, it is difficult to assess whether the reported effects were due to the incorporated or free fatty acids. Moreover, in studies where the parent compounds LA or ALA were used, it was not possible to untangle the effects of the parent compounds, from that of their metabolites.
Three human cell lines namely, acute monocytic leukemia (THP-1) colon adenocarcinoma (HT29) and hepatocellular carcinoma (HepG2) have been used in the present research. Initially, it was tested their ability to convert ALA to EPA and DHA, as well as to synthesise AA from LA. In addition, it was evaluated the incorporation of preformed EPA, DHA and AA. Subsequently, it was elucidated whether the synthesis of EPA, DHA, and AA was enhanced by all-trans retinol (vitamin A) or its analogs 9-cis (9-cis RA) and all trans retinoic (ATRA) acids. Additionally, it was determined whether the expression of FADS2, FADS1, ACOX1 and PPARA genes coding for Δ6 (D6D), Δ5 (D5D) desaturases, AcylCoA oxidase and peroxisome proliferator activated receptor α (PPAR α) are expressed in THP-1 and HT29 cells and are altered by omega-3 and -6 PUFA. Finally, it was elucidated if changes in membrane fatty acid composition affected cellular signalling with a specific focus on insulin transduction.
It was concluded that THP-1, HT29 and HepG2 cells were able to incorporate omega-3 and -6 PUFA but had different abilities to convert ALA or LA to their metabolites. THP-1 cells were able to convert ALA to docosapentaenoic acid omega-3 (DPA) and incorporate it in both the CPG and EPG fractions. LA treatment increased AA percentage in the CPG. In the CPG and EPG, exposure of HT29 cells to LA increased the percentage of AA. In HepG2 cells, LA incubation marginally increased adrenic acid (ADA) in the EPG. In all cells EPA treatments increased the level of DPA in EPG and CPG.
Retinoids modified the utilisation of omega-3 and -6 PUFA. In THP-1 cells the combination of retinoids and ALA increased ALA percentage compared with ALA incubation and it reduced the production of EPA. This observation suggested inhibition of ALA conversion and promotion of ALA accumulation. On the contrary, in HT29 cells, retinoids plus ALA showed diminished accumulation of ALA.
In THP-1 cells, retinol or ATRA combined with LA increased the accumulation of LA compared to LA alone treatments. In HT29 cells, it was interesting to notice that the sole utilisation of retinoids increased AA levels.
Additionally, the data showed that omega-3 and -6 PUFA differentially altered the expression of FADS1, FADS2, ACOX1 and PPARA genes. Different outcomes were reported for THP-1 and HT29 cells. ALA and EPA treatments lowered the expression of FADS2. On the contrary, in HT29, FADS2 expression was enhanced by ALA treatment. In THP-1 cells, LA decreased ACOX1 and FADS1, whilst AA decreased the level of FADS2. EPA incubation enhanced the level of ACOX1 in HT29 cells. The expression of PPARA was increased by EPA in THP-1 cells, and decreased in HT29 after EPA, DHA, LA and AA incubations.
Finally, the data showed that increased levels of LA or AA in HT29 cells enhanced insulin-stimulated phosphorylation of the AKT protein.
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