Competition between 24:5n-3 and ALA for Δ6 desaturase may limit the accumulation of DHA in HepG2 cell membranes

Abstract

The use of #6 desaturase (D6D) twice in the conversion of a-linolenic acid (ALA; 18:3n-3) to docosahexaenoic acid (DHA; suggests that this enzyme may play a key regulatory role in the synthesis and accumulation of DHA from ALA. We examined this using an in vitro model of fatty acid metabolism to measure the accumulation of the long-chain metabolites of ALA in HepG2 cell phospholipids. The accumulation of ALA, eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-3), and 24:5n-3 in cell phospholipids was linearly related to the concentration of supplemented ALA over the range tested (1.8-72 mM). The accumulation of the post-D6D products of 22:5n-3, 24:6n-3 and DHA, in cell phospholipids was saturated at concentrations of .18 mM ALA. Supplementation of HepG2 cells with preformed DHA revealed that, although the accumulation of DHA in cell phospholipids approached saturation, the level of DHA in cell phospholipids was significantly greater compared with the accumulation of DHA from ALA, indicating that the accumulation of DHA from ALA was not limited by incorporation. The parallel pattern of accumulation of 24:6n-3 and DHA in response to increasing concentrations of ALA suggests that the competition between 24:5n-3 and ALA for D6D may contribute to the limited accumulation of DHA in cell membranes. The synthesis of long-chain polyunsaturated fatty acids (LCPUFAs) from a-linolenic acid (ALA; 18:3n-3) and linoleic acid (LA; 18:2n-6) is well documented, yet regulation of the pathway, particularly the regulation of the conversion of ALA to docosahexaenoic acid (DHA; 22:6n-3), remains a focus of investigation. This is attributable to the observation that increased dietary intakes of ALA in animals and humans result in an increased level of eicosapentaenoic acid (EPA; 20:5n-3) but little or no change in the level of DHA in tissues or plasma (1-3). Direct measurement of fatty acid synthesis in humans using labeled ALA has demonstrated the conversion of ALA to EPA and docosapentaenoic acid (DPA; 22:5n-3) with limited conversion to DHA (1, 4-6). There are several plausible explanations for the disparity between the intake of ALA and its conversion to DHA in vivo. Ingested ALA has several metabolic fates, including b-oxidation, carbon recycling, conversion to LCPUFA, and direct incorporation into structural lipids (7). The balance between these metabolic fates influences the accumulation of DHA from ALA in tissues. Additionally, both n-3 and n-6 fatty acids use common enzymes in the synthesis of fatty acids; therefore, the competition between n-3 and n-6 fatty acid substrates for these enzymes is an important determinant of DHA synthesis. ALA and LA are both substrates for D6 desaturase (D6D). When both of these substrates are present, there is competition for active sites on D6D, with ALA being the preferred substrate (8). The affinity of D6D for ALA is approximately two to three times that of LA (9). The competition between substrates for D6D also extends to the 24 carbon fatty acids (24:5n-3 and 24:4n-6) a...

Barramundi (Lates calcarifer) desaturase with Δ6/Δ8 dual activities

“…Increasing ALA concentrations in HepG2 cells resulted in increasing and then decreasing DHA production, suggesting competition between n-3 substrates for Δ6 desaturase at the higher concentrations of ALA (Portolesi et al 2007). …”

Linoleic and α-linolenic acid as precursor and inhibitor for the synthesis of long-chain polyunsaturated fatty acids in liver and brain of growing pigs

“…This finding suggests a rate-limiting enzyme downstream of ELOVL2. As D6 desaturase has a higher affinity for ALA than for c24:5 n-3 (D' Andrea et al, 2002;Portolesi et al, 2007), it is conceivable that D6 desaturation of c24:5 n-3 is inhibited when ALA is abundant, preventing DHA synthesis. We would suggest that in the liver of piglets, D6 desaturase rather than ELOVL2 is the rate-limiting enzyme downstream of EPA in the biosynthesis of DHA.…”

Fat and Fatty Acid Terminology, Methods of Analysis and Fat Digestion and Metabolism: A Background Review Paper

“…Most likely, both 24: 5n-3 and 24: 4n-6 are in direct competition with LA and ALA for access to the ⌬ 6 desaturase. These competitions further limit the formation of DHA in tissues [Portolesi et al, 2007].…”