Tissue-specific, nutritional, and developmental regulation of rat fatty acid elongases

Abstract

Of the six fatty acid elongase (Elovl) subtypes expressed in mammals, adult rat liver expresses four subtypes: Elovl-5 > Elovl-1 = Elovl-2 = Elovl-6. Overnight starvation and fish oil-enriched diets repressed hepatic elongase activity in livers of adult male rats. Diet-induced changes in elongase activity correlate with Elovl-5 and Elovl-6 mRNA abundance. Adult rats fed the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist WY14,643 have increased hepatic elongase activity, Elovl-1, Elovl-5, Elovl-6, Delta5, Delta6, and Delta9 desaturase mRNA abundance, and mead acid (20:3,n-9) content. PPARalpha agonists affect both fatty acid elongation and desaturation pathways leading to changes in hepatic lipid composition. Elovl activity is low in fetal liver but increases significantly after birth. Developmental changes in hepatic elongase activity paralleled the postnatal induction of Elovl-5 mRNA and mRNAs encoding the PPARalpha-regulated transcripts, Delta5 and Delta6 desaturase, and cytochrome P450 4A. In contrast, Elovl-6, Delta9 desaturase, and FAS mRNA abundance paralleled changes in hepatic sterol regulatory element binding protein 1c (SREBP-1c) nuclear content. SREBP-1c is present in fetal liver nuclei, absent from nuclei immediately after birth, and reappears in nuclei at weaning, 21 days postpartum. In conclusion, changes in Elovl-5 expression may account for much of the nutritional and developmental control of fatty acid elongation activity in the rat liver.

Fig. 1

Tissue-specific expression of rat fatty acid elongases (Elovls). RNA was extracted from rat liver, lung, brain, brown adipose tissue (BAT), kidney, skin, and heart. These tissues were obtained from a 60 day old male rat maintained on a Harlan-Teklad chow diet ad libitum. Elovl-1, Elovl-3, and Elovl-5 mRNAs were detected by Northern blot analysis (NB) using cDNAs cloned as described in Materials and Methods. Elovl-2 and Elovl-6 were detected by RT-PCR (35 cycles) (PCR).

Fig. 2

Effect of diet on rat hepatic elongase and desaturase expression. Male Sprague-Dawley rats (50 days of age) were meal-fed a high carbohydrate (HiCHO) diet supplemented with olive oil (Olive; 10%, w/w), fish oil (Fish; 10%, w/w), or olive oil plus WY14,643 (WY14,643; 0.1%, w/w) for 7 days. Fasted animals were meal-fed the HiCHO-olive oil diet as described above but were fasted for 24 h before euthanasia. A: Northern blots of elongase and desaturase mRNAs. A rat liver standard (S) derived from a 90 day old rat fed Harlan-Teklad chow was included in all Northern blots for Elovl-1, Elovl-2, Elovl-5, and Elovl-6 and Δ⁵, Δ⁶, and Δ⁹ desaturase mRNA measurements. Single transcripts were detected for each mRNA, except Elovl-6. Two transcripts have been reported for Elovl-6 (10). In all cases, the size of the mRNA corresponded with the reported size of the transcript. A representative ethidium bromide stain of 18S and 28S mRNA documents equal loading of the RNA samples. Elovl-3 was not detected in any treatment (not shown). B: Fold changes in Elovl, fatty acid desaturase (D), and FAS mRNA abundance after dietary challenge. Top panel: Effect of fasting and refeeding on mRNA expression. Results are expressed as means ± SD; n = 3/animals/group. * P ≤ 0.05, fasted versus fed. Middle panel: Effect of fish oil feeding on mRNA expression. Results are expressed as means ± SD; n = 6/group. * P ≤ 0.05, olive oil versus fish oil. Bottom panel: Effect of WY14,643 feeding on mRNA expression. Results are expressed as means ± SD; n = 6/group. * P ≤ 0.05, olive oil versus olive oil + WY14,643. The dashed lined in each panel represents a fold change of 1.0.

Fig. 3

Effect of diet on hepatic Elovl activity. In vitro assays for elongase activity used microsomes isolated from livers of rats fasted or fed a HiCHO diet containing olive oil, fish oil, or WY14,643 as described in Materials and Methods. Separate reactions were run using 16:0-CoA, 18:0-CoA, 18:1,n-9-CoA, 20:0-CoA, 20:4,n-6-CoA, 22:0-CoA, and 24:0-CoA as substrates. Results are expressed as elongase activity units (nmol [¹⁴C]malo-nyl-CoA incorporated/mg protein), means ± SD; n = 3/group. * P ≤ 0.05, olive oil-fed rats versus fasted, fish oil fed-, WY14,643 fed-, or Harlan-Teklad chow-fed rats.

Fig. 4

Effect of diet on plasma and liver mead acid levels. Total lipids extracted from rat liver and plasma of animals fed olive oil, fish oil, or WY14,643 were saponified, separated by reversed-phase HPLC, and quantified (see Materials and Methods). The identities of all fatty acids were confirmed by gas chromatography-mass spectrometry. Levels of mead acid (20:3,n-9) and arachidonic acid (20: 4,n-6) were quantified and the results are presented as mol% of the total saponified lipid fraction (means ± SD; n = 3/animals/group). The mol% of 20:3,n-9 in olive oil-fed versus fish oil-fed animals and in olive oil-fed versus olive oil + WY14,643-fed animals was significantly different (P < 0.01).

Fig. 5

Developmental regulation of rat hepatic sterol regulatory element binding protein 1 (SREBP-1) and SREBP-2 nuclear content and Elovl, fatty acid desaturase, and fatty acid synthase mRNA abundance. A: Immunoblot. Nuclear proteins isolated from livers at 18 days after coitus (Fetal) and 1, 7, 15, and 30 days postpartum were prepared, separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies for SREBP-1 and SREBP-2 (35). The inclusion of SREBP-2 shows that hepatic SREBP-1 and SREBP-2 nuclear contents are regulated differently during development. B: mRNA abundance. Rat liver mRNAs from 18 days after coitus (Fetal) and 10 and 30 days postpartum were used to quantify levels of Elovls (Elovl-1, Elovl-2, Elovl-5, and Elovl-6), fatty acid desatu-rases (Δ⁵ D, Δ⁶ D, and Δ⁹ D), and FAS. The results are represented as relative RNA abundance and are normalized to the level of expression in a 90 day old male rat (means ± SD; n = 3/group). * P ≤ 0.05, fetal versus 10 or 30 days.

Fig. 6

Rapid induction of hepatic Elovl-5 and cytochrome P450 4A (CYP4A) mRNA after parturition. Immunoblot (IB) CYP4A and PPARα (peroxisome proliferator-activated receptor α): Nuclear (PPARα) or microsomal (CYP4A) proteins were isolated from livers at 18 days after coitus (Fetal) and 3, 7, 10, 15, 18, 21, and 30 days postpartum. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies for PPARα or CYP4A (35). Northern blot (NB) CYP4A: Total RNA was extracted from rat livers at 18 days after coitus (Fetal) and 1, 7, 15, 21, and 30 days postpartum (n = 3/group). S (standard), RNA from 90 day old rat liver. RNA electrophoretically separated and transferred to nitrocellulose was probed for CYP4A (50). Northern blot (NB) Elovl-5: Total RNA was extracted from livers of fetal rats and from male rats 3, 7, 10, 15, 18, 21, and 90 days old. RNA electrophoretically separated and transferred to nitrocellulose was probed for Elovl-5.

Fig. 7

Developmental regulation of hepatic Elovl activity. In vitro assays for elongase activity used microsomes isolated from livers of fetal rats (18 days after coitus) and 10, 30, and 90 days postpartum. Separate reactions were run using 16:0-CoA, 18:0-CoA, 18:1,n-9-CoA, 20:0-CoA, 20:4,n-6-CoA, 22:0-CoA, and 24: 0-CoA as substrates. Results are expressed as elongase activity units (nmol [¹⁴C]malonyl-CoA incorporated/mg protein), means ± SD; n = 3/group. Elongase activity in fetal liver was significantly lower than in postpartum liver for all substrates examined (P ≤ 0.05).