ABSTRACT
Desaturase Activities and Gene Expression. The ∆6 n-6 and ∆6 n-3 desaturase activities were measured according to Narce et al. (12) using liver microsomal membrane preparations (13). The expression of ∆6-desaturase (∆6D) was determined by Northern blotting, after tissue mRNA extraction. Total RNA was isolated from frozen liver according to Chomczynski and Sacchi (14), and quantified by ultraviolet (UV) light absorption at 260 nm. The integrity of the RNA used for the RNAse protection analysis was verified by fractionating 5 µg of total hepatocyte RNA on a 1% agarose gel containing 6.7% formaldehyde and visualizing RNA by ethidium bromide staining. Electrophoresis and Northern blotting were performed as follows: 50 µg of total RNA from each liver was denatured in 50% formamide, 17% formaldehyde, 3-(N-morpholino) propoanesulfonic acid (MOPS) buffer (20 mM MOPS, 5mM sodium acetate, pH 7.0, 1mM EDTA) for 10 min at 65°C, and run on a denaturing agarose gel in MOPS buffer. Transfer onto nylon membranes (HybondN, Amersham, Les Ulis, France) was achieved by capillary blotting using 10 XSSC buffer (1.5 M NaCl, 0.15 M trisodium citrate, pH 7.4). Membranes were then air-dried and irradiated with UV light at 254 nm in a spectrolinker XL-1000 UV crosslinker (Spectronics) with an energy of 1.2 × 10-3 J/cm2. The mRNAs were then hybridized with human DNA probes. Full-length cDNA clones were used for ∆6D and 18S. Inserts from these different clones were purified using Qiaquick Spin Columns (Qiagen, Courtaboeuf, France) and then 32P-radiolabeled using a random priming labeling kit (multiprime DNA labeling kit, RPN 1601 Y,
Introduction Among factors regulating polyunsaturated fatty acid (PUFA) biosynthesis, it has been shown that dietary fatty acids play a key role. Their influence is dependent on their family (n-6 or n-3), their chain length, and their number of double bounds (1-3). Their effects on desaturase activities remain controversial. For example, Hoy et al. (4) showed in rats that low dietary supplementations with oils rich in n-3 PUFA had no effect on desaturation, whereas Blond et al. (5) demonstrated that dietary 18:3n-3 (ALA) inhibited both ∆6 and ∆5 desaturations. Linseed oil has been shown to inhibit ∆6 (1), but increase ∆5 desaturation (2). On the contrary, Christiansen et al. (6) observed an increase of ∆6 and ∆5 desaturase activities with such a diet. More recent studies have shown that dietary n-6 or n-3 PUFA decreased hepatic mRNA of ∆6 desaturase by ~80% and that of ∆5 desaturase by ~60% (7). Nevertheless, the control diet used in those investigations was a high-glucose/low-fat diet, which stimulates desaturation. In sum, discrepancies exist among the data on regulation of PUFA biosynthesis by dietary fatty acids; these discrepancies result from differences in control diets (fat-free, high-glucose, high-fat), the origin or doses of administered fats (oils, fatty acid mixtures, purified fatty acids), the age of the animals, or the duration of the treatments.
