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Endocrinology, doi:10.1210/en.2007-1402
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Endocrinology Vol. 149, No. 6 3158-3166
Copyright © 2008 by The Endocrine Society

Steroid Hormones Control Circadian Elovl3 Expression in Mouse Liver

Annelie Brolinson, Stéphane Fourcade, Andreas Jakobsson, Aurora Pujol and Anders Jacobsson

The Wenner-Gren Institute (A.B., A.Jac., A.Jak.), The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden; Centre de Genètica Mèdica i Molecular (S.F., A.P.), Institut d'Investigació Biomèdica de Bellvitge; Institut de Neuropatologia de Bellvitge (S.F., A.P.), and Institut d'Investigació Biomèdica de Bellvitge and Universitat de Barcelona, Barcelona 08028, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain; and Catalan Institution of Research and Advanced Studies, ICREA (A.P.), 08010 Barcelona, Spain

Address all correspondence and requests for reprints to: Anders Jacobsson, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-10691 Stockholm, Sweden. E-mail: anders.jacobsson{at}wgi.su.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Elovl3 gene belongs to the Elovl gene family, which encodes for enzymes involved in the elongation of very long chain fatty acids. The recognized role for the enzyme is to control the elongation of saturated and monounsaturated fatty acids up to 24 carbons in length. Elovl3 was originally identified as a highly expressed gene in brown adipose tissue on cold exposure. Here we show that hepatic Elovl3 mRNA expression follows a distinct diurnal rhythm exclusively in mature male mice, with a sharp increase early in the morning Zeitgeber time (ZT) 20, peaks around ZT2, and is back to basal level at the end of the light period at ZT10. In female mice and sexually immature male mice, the Elovl3 expression was constantly low. Fasting and refeeding mice with chow or high-fat diet did not alter the Elovl3 mRNA levels. However, animals that were exclusively fed during the day for 9 d displayed an inverted expression profile. In addition, we show that Elovl3 expression is transcriptionally controlled and significantly induced by the exposure of the synthetic glucocorticoid dexamethasone. Taken together, these data suggest that Elovl3 expression in mouse liver is under strict diurnal control by circulating steroid hormones such as glucocorticoids and androgens. Finally, Elovl3 expression was found to be elevated in peroxisomal transporter ATP-binding cassette, subfamily D(ALD), member 2 ablated mice and suppressed in ATP-binding cassette subfamily D(ALD) member 2 overexpressing mice, implying a tight cross talk between very long chain fatty acid synthesis and peroxisomal fatty acid oxidation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ABILITY TO synthesize very long chain fatty acids (VLCFAs) is a ubiquitous system found in most cell types. However, VLCFAs seldom occur unesterified. Instead, they are joined in ester, ether, or amide linkage to a broad variety of different lipid species. VLCFAs are most commonly found as building blocks in ceramides and sphingolipids (1). In addition, they are important constituents of glycerophospholipids, triglycerides, and sterol and wax esters (2, 3). The physiological significance of VLCFAs are indisputable, and there are a number of mammalian disorders that are known to be related to abnormal levels of VLCFAs such as myelin deficiency and peroxisomal disorders involving the adrenal cortex and the nervous system (4, 5).

Formation of VLCFAs mainly occurs in the endoplasmic reticulum (6) and is controlled by at least six different elongation of VLCFA (ELOVL) enzymes that are believed to exert substrate-specific elongation with respect to fatty acid length and unsaturation (7, 8, 9). ELOVL6 regulates the synthesis of C18:0 and 18:1, whereas ELOVL1 and ELOVL3 are suggested to regulate the synthesis of saturated and monounsaturated fatty acids up to C26 and C24, respectively. ELOVL2, -4, and -5 use polyunsaturated fatty acid as substrates. All six Elovl genes show a diverse tissue-specific expression pattern, indicating a unique role for different VLCFAs in different cell types. Nevertheless, there are hardly any reports on how the levels of VLCFAs are controlled in any organism or the significance of endogenous synthesized VLCFAs vs. VLCFAs taken up from the diet.

So far, no data exist on posttranscriptional control of any ELOVL protein or fatty acid synthase (FAS). Available data rather favor that the activity is controlled by transcriptional regulation. We know from expression studies in mouse brown adipose tissue and primary cell cultures that Elovl3 expression is under a synergistic control of glucocorticoids and noradrenalin (10). Recent studies show that peroxisome proliferator-activated receptors (PPARs) are, in conjunction with norepinephrine and glucocorticoids, very potent inducers of Elovl3 expression (11, 12). On the other hand, we found that the stimulation of Elovl3 expression was independent of activation of the lipogenic factors liver X receptor (LXR) and sterol regulatory element-binding protein (SREBP)-1, which normally induce lipogenic genes including acetyl-CoA carboxylase (ACC), FAS, and Elovl6 (11, 13, 14, 15).

Elovl3-ablated mice show impaired formation of triglycerides and lipid droplets in both skin and brown adipose tissue (16, 17). Data imply that activation of Elovl3 expression is linked to increased FA oxidation and that the function for the enzyme in brown adipocytes during cold exposure is to replenish the intracellular pool of fatty acids to maintain lipid homeostasis.

Liver is a highly metabolic organ responsible for uptake, storage, and release of lipids into the circulation to meet the energy demands of the body. To maintain proper lipid homeostasis, the regulation of lipid synthesis is highly coordinated with fatty acid and glucose uptake from the diet (18).

Several of the genes involved in fatty acid synthesis such as ACC and FAS show a diurnal variation of their expression, which is strongly correlated with food intake (19). Studies also imply that the diurnal expression of ACC and FAS is controlled by the PPAR{alpha} (20). In brown adipose tissue, cold-induced Elovl3 expression is significantly reduced in PPAR{alpha}–/– mice (11).

Recently Anzulovich et al. (21) showed that, in liver, Elovl3 expression follows a circadian pattern, which is perturbed in mutant CLOCK mice. Elovl3 promoter analyses revealed that RevErba repressed the expression, whereas SREBP-1 had a stimulatory effect.

Here we show that hepatic Elovl3 expression is under a strong diurnal control exclusively in sexual mature male mice, which is independent of PPAR{alpha}. The expression is not affected by fasting or daily food intake but rather controlled by a circadian release of glucocorticoids and gender-specific steroids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal experiments
NMRI male mice (obtained from B&K Universal, Stockholm, Sweden), between 8 and 17 wk old, and 10-wk-old castrated mice were used except for the gender- and age-specific experiments in which 4-wk-old male mice and 8-wk-old female mice were used. Twelve-week-old ovariectomized NMRI mice were purchased from Taconic (Lille Skensved, Denmark). Eight- to 10-wk-old PPAR{alpha}-ablated mice, backcrossed on DBA background, were used (kind gift from Dr. F. Gonzales) (22). As control mice, age-matched mice from this backcross, which were bred under the same conditions as the PPAR{alpha}-ablated mice, were used. Mice overexpressing or lacking the ATP-binding cassette subfamily D(ALD) member (ABCD) 2 protein have already been described (23, 24). In this study we used 6-wk-old male mice of a mixed genetic background (75% C57BL/6J and 25% 129Sv).

All mice were housed in 22 C and maintained on a 12-h light, 12-h dark cycle [lights on at Zeitgeber time (ZT) 0]. Animals were fed ad libitum, unless otherwise stated, with rat and mouse standard diet no.1 (BeeKay Feeds; B&K Universal, Stockholm, Sweden) and had free access to water. In feeding experiments, male mice were food deprived for 17 h, starting at ZT10 (1800 h). The animals were either refed with chow diet or high-fat diet, (45 kcal% fat) (D12451; Research Diets, New Brunswick NJ). For the conditional feeding experiment, NMRI mice received food only during the day (ZT0-ZT12) for 9 d, and the control mice were fed ad libitum. For actinomycin treatment. mice were injected ip with either actinomycin D (A5156; Sigma, St. Louis, MO), 0.025 mg/mouse, or sterilized NaCl-solution (control group). One group of mice were injected at ZT22 (0600 h), and the other group of mice were injected at ZT2.5 (1030 h). Both groups were killed 3.5 h after injection. For dexamethasone treatment, mice were injected ip with dexamethasone (Vetranal, 46165; Sigma), 0.1 mg/ mouse, or vehicle at ZT2. The livers were dissected at ZT10 and total RNA was isolated. All mice were killed with CO2 and the livers were dissected and frozen in liquid nitrogen. Before experiments the animals were accustomed to the environment for at least 1 wk.

All studies were carried out with ethical permission from the Animal Ethics Board in Stockholm, Sweden.

RNA analysis
RNA was isolated with Ultraspec RNA isolation solution (Biosite, San Diego, CA), and total RNA was isolated according to the manufacturer’s procedure. Ten or 15 µm of total RNA were separated by electrophoresis in a 1.2% agarose-formaldehyde gel containing ethidium bromide and blotted to Hybond-N membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Prehybridization and hybridization were carried out overnight at 42 C in 50% (vol/vol) formamide, 5x saline sodium citrate (SSC), 5x Denhart’s solution (2% Ficoll, 2% BSA, and 2% polyvinylpyrolidone), 0.5% sodium dodecyl sulfate (SDS), 50 mM sodium phosphate (pH 6.5), and 0.1 mg/ml degraded herring sperm DNA. Membranes were washed twice in 2x SSC and 0.1% SDS at room temperature for 15 min and once in 0.1x SSC and 0.1% SDS at 42 C for 20 min. cDNA fragments, identified by BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), and verified by sequencing for Elovl1 and Elovl3 (25), 11β-Hsd1 [IMAGE EST clone GenInfo Identifier (gi) 14605089 (I.M.A.G.E. Consortium, Geneservice Ltd., Cambridge, UK)], Elovl6 (gi: 15715540), LXR{alpha} (gi: 15215049), FAS (gi: 11366366), PPAR{alpha} (gi: 11271556), were [32P]dCTP-labeled by using random-primed DNA labeling kit Ready to go DNA labeling beads (Amersham, Buckinghamshire, UK) Aylesbury, UK) and used as probes. Blots were exposed to a PhosphoImager screen analyzed in a FLA-3000 reader (Fuji, Tokyo, Japan). 18S rRNA was detected using a 473-nm laser in the FLA reader and used for normalization. RNA levels were quantified using Fuji Film Science Lab software (Tokyo, Japan), and statistics were calculated in GraphPad PRISM (San Diego, CA). Statistical differences were calculated with unpaired t test (P < 0.05).

For reverse transcription (RT) and quantitative PCR, 1 µg total RNA was mixed with 4 µl random hexamers (0.05 µg/µl) and diethylpyrocarbonate water and incubated 5 min on 70 C and then chilled on ice and briefly centrifuged.

Subsequently 4 µl of RT reaction buffer (5 times), 1 µl RNase out (40 U), and 2 µl deoxynucleotide triphosphates (10 mM) were added to the mixture and incubated for 5 min at 25 C. One microliter of Moloney murine leukemia virus RT enzyme (200 U/µl) was added, and the mixture was incubated for 10 min at 25 C followed by an incubation for 60 min at 42 C. The reaction was inactivated by incubation for 10 min at 70 C. RT-quantitative PCR aliquots with 2 µl of the sample cDNA were mixed with TaqMan master mix (Applied Biosystems Inc., Foster City, CA), 18S, or Elovl3 probes and double-distilled H2O to a final volume of 24 µl/well and were measured in triplicate for each sample. Two animals were used in each group. Assay-on-demand gene expression products, purchased from Applied Biosystems, were used (Elovl3-Mm00468164_m1 and 18S-HS99999901_S1). Expression was analyzed in a Applied Biosystems Prism 7000 sequence detection system.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Strong diurnal expression of Elovl3 in liver
We earlier demonstrated a differential regulation between Elovl3 and Fas and two other fatty acid elongases, Elovl1 and Elovl6, in brown adipose tissue by the lipogenic transcription factors LXR{alpha}, SREBP-1, and PPAR{alpha} (11). As seen in Fig. 1AGo, in contrast to most other genes analyzed including Fas, LXR{alpha}, Elovl1, and Elovl6, a dramatic change was seen for the Elovl3 expression in liver during the day. There was a distinct increase in the amount of Elovl3 mRNA during the end of the dark phase at ZT20 (0400 h) and a dramatic decrease after ZT 8 (1600 h) at the end of the light period. The highest Elovl3 expression was seen at ZT2 (1000 h) in the beginning of the light period (Fig. 1BGo). In contrast, the mRNA levels of Elovl1, which, similar to Elovl3, is also proposed to elongate saturated and monounsaturated fatty acids, did not show any strong diurnal rhythm (Fig. 1CGo). Interestingly, the diurnal expression pattern for Elovl3 was different from the pattern earlier reported for other lipogenic genes such as Fas, SREBP-1, or PPAR{alpha} (19, 26). In addition, we did not detect any zonal differences in mRNA expression of Elovl3 within the liver (data not shown).


Figure 1
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  		FIG. 1. Diurnal variation of Elovl3 expression in mouse liver tissue. Examination of gene expression around the day in liver by Northern blotting. mRNA levels of the fatty acid elongases Elovl3, Elovl1, and Elovl6 and Fas, LXR, and PPAR{alpha}, the typical marker of fatty acid β-oxidation, Acyl-CoA oxidase 1 (Aox), and the main regulatory enzyme in glucocorticoid metabolism, 11β-HSD (Hsd-1) in 8-wk-old male mouse liver tissue were isolated at indicated times (A). Elovl3 (B) and Elovl1 (C) mRNA levels were analyzed by Northern blots of liver tissues taken from mice at indicated times. Results shown are the means ± SEM of values from three animals (15 µg RNA each) at each time point.

 
Neither starvation nor refeeding but feeding schedule control Elovl3 expression in liver
The expression of lipogenic genes involved in FAS and lipid storage in liver are shown to adapt rapidly to times of food intake (27). For example, the expression of Fas, Acc, sterol CoA desaturase-1 (Scd1), and the fatty acid elongase Elovl6, which control the elongation of C16:0 and C16:1 to C18:0 and C18:1, respectively, are repressed during starvation and induced during refeeding (28, 29, 30). To elucidate the effects of food intake on Elovl3 expression, 8-wk-old male mice were food deprived for 17 h, starting at ZT10 (1800 h). The animals were either refed with chow diet or a high-fat diet. As seen in Fig. 2AGo, the diurnal expression of Elovl3 was not altered when the animals were fasted for 17 h or after refeeding. In addition, no difference was seen between animals refed chow or high-fat diet (Fig. 2AGo).


Figure 2
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  		FIG. 2. Feeding control of Elovl3 expression. Eight-week-old male mice were food deprived for 17 h, starting at ZT10. The mice were refed with either standard chow (open squares) or high-fat diet (filled squares) from ZT3 in the morning. Elovl3 mRNA levels were measured at indicated times (A). Mice were exclusively fed during the day (ZT0-ZT12) for 9 d and Elovl3 and Elovl1 mRNA levels were analyzed (B and C). Results shown are the means ± SEM of values from three animals (10 µg RNA each) at each time point. *, Significance with P < 0.05.

 
To further elucidate the diurnal expression of Elovl3 in liver, mice were exclusively fed during the day (ZT0-ZT12) for 9 d, and RNA levels were analyzed at different time points. As seen in Fig. 2BGo, there was a significant change in the Elovl3 expression pattern in animals fed only during the day. The Elovl3 expression reached the highest level during the beginning of the dark period, showing that the diurnal variation was shifted about 12 h, compared with mice fed ad libitum. In addition, the basal values were slightly higher in the food-restricted animals. No significant effect was seen on Elovl1 expression in conditional fed animals (Fig. 2CGo). These data demonstrate that Elovl3 is under differential transcriptional regulation and concomitantly has a different functional role in liver lipid metabolism, compared with traditional lipogenic genes controlling fatty acid synthesis and energy storage.

Transcriptional control of circadian Elovl3 expression
Because the mRNA level of Elovl3 was dramatically reduced at the end of the light period, we asked whether this was due to a change in transcription activity or a decreased half-life of the transcript or both. To investigate this, transcription was blocked by injecting animals with actinomycin D, and mRNA levels were analyzed 3.5 h later, both during the induction phase (ZT22-ZT1.5) when Elovl3 mRNA level normally is increased and during the reduction phase (ZT2.5-ZT6) when the mRNA level normally is decreased. During the induction phase, the Elovl3 mRNA level was further increased in saline injected mice (Fig. 3AGo). However, in actinomycin-injected mice, the mRNA level was decreased to about 60% of starting level. In contrast, during the reduction phase, there was no statistical difference between actinomycin- and saline-injected mice (Fig. 3BGo). The mRNA levels were reduced to about 50% in both groups. The mRNA levels of Elovl1 did not change significantly at any time (Fig. 3CGo). This implies that transcription is the major regulator of the Elovl3 mRNA level in liver and that the estimated half-life of the transcript is about 3 h, which does not significantly change during the day. Interestingly, this is in sharp contrast to what we have seen in brown adipose tissue in which the level of Elovl3 transcript is controlled by both transcription and mRNA stability and with an estimated half-life of about 20 h (12).


Figure 3
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  		FIG. 3. Transcriptional control of Elovl3 mRNA levels. Eight-week-old male mice were injected with actinomycin (Act) or saline (NaCl) at ZT22 or ZT2.5, respectively. The livers were isolated 3.5 h after injection. Elovl3 mRNA levels were analyzed by Northern blot at ZT1.5 (A) and ZT6 (B), respectively. Elovl1 mRNA levels were analyzed at ZT6 (C). Results are means ± SEM of values from three animals (10 µg RNA each) at each time point.

 
Glucocorticoid control of Elovl3 expression
We have earlier shown that glucocorticoids together with norepinephrine synergistically induce Elovl3 expression in brown adipose tissue (10, 11). The glucocorticoid receptor, which belongs to the nuclear hormone receptor family, plays a central role in the circadian control of fatty acid and carbohydrate metabolism (31, 32). Glucocorticoids are predominantly secreted from the adrenal cortex in daily cycles, and in rodents, the hormone levels peak during late day or early evening (33). When taken up by the cell, a hormone-receptor complex is formed in the cytosol and translocated into the nucleus in which it functions as a transcription factor to control gene expression (34).

Daytime feeding for a period of time alters the daily glucocorticoid secretion in nocturnal animals independently of the light cycle (33). If the circadian Elovl3 expression in liver is dependent on the level of glucocorticoids, the latency period is approximately 8 h. To test this, the synthetic glucocorticoid receptor agonist dexamethasone, which has been shown to be a potent phase-shifting agent in peripheral tissues (35), was injected ip into mice at ZT2 when serum levels of glucocorticoids normally are low. Eight hours later the mRNA levels of Elovl3 in liver were analyzed. As seen in Fig. 4Go, A and B, there was an increased level of Elovl3 but not of Elovl1 transcript at ZT10 in the animals injected with dexamethasone, compared with the animals injected with sodium chloride, which suggest that the diurnal control of Elovl3 expression is dependent on the level of glucocorticoids in the serum. In a recent study (12) on cultured brown adipocytes, we suggested that glucocorticoids are required for enhanced Elovl3 mRNA stability, which is a mechanism that we cannot rule out and also takes place in liver. Injection of norepinephrine, which has been shown to induce Elovl3 expression in brown adipose tissue, had no effect on Elovl3 expression in liver (not shown).


Figure 4
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  		FIG. 4. Glucocorticoid and transcriptional control of Elovl3 mRNA levels. The synthetic glucocorticoid dexamethasone (D) or vehicle (V) was injected into 8-wk-old male mice at ZT2 and Elovl3 (A) and Elovl1 (B) mRNA levels, respectively, were analyzed at ZT10. Results shown are the means ± SEM of values from three animals (10 µg RNA) at each time point. *, Significance with P < 0.05.

 
Gender-specific control of Elovl3 expression
Because glucocorticoid species are known to differ between genders, with females having higher serum levels (36), we compared the diurnal variation of Elovl3 expression in male and female mice. To our surprise, the Elovl3 mRNA was almost undetectable in female mice at any time point of the day (Fig. 5AGo). In addition, there was no visible expression of Elovl3 in sexually immature 4-wk-old male mice (Fig. 5AGo). However, by performing quantitative PCR analysis, we identified significant amounts of Elovl3 transcript in both female and immature male mice (not shown). In contrast, Elovl1 was expressed at a similar level regardless of sex and age (Fig. 5AGo). This suggests that androgens are essential for the diurnal Elovl3 expression in liver. To confirm this, we analyzed Elovl3 expression in castrated, 10-wk-old male mice. As seen in Fig. 5BGo, there was undetectable expression in these mice at ZT2 when the expression is normally high in control mice. In contrast, there was no effect on Elovl3 expression in ovariectomized mice (not shown), suggesting that if estrogen is acting as a suppressor of Elovl3 expression, female mice require additional signaling to induce diurnal expression of Elovl3.


Figure 5
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  		FIG. 5. Diurnal Elovl3 expression exclusively in sexually mature male mice. Representative Northern blot of Elovl3 and Elovl1 mRNA levels in liver (10 µg RNA) from 4- and 8-wk-old male and female mice (A). Elovl3 expression was analyzed in 10-wk-old castrated and wild-type male mice (B).

 
PPAR{alpha}-independent regulation of circadian Elovl3 expression
Hepatocytes assimilate carbohydrates and lipid signals in part through the transcription factor PPAR{alpha}. Although PPAR{alpha} is a promoting actor of catabolic processes, it has also been shown to be involved in de novo lipogenesis in liver. For example, the diurnal rhythms for ACC and FAS are abolished in PPAR{alpha}–/–mice (20). We have earlier shown that the induced Elovl3 expression in brown adipocytes, in both animals and primary cell cultures, was significantly reduced in PPAR{alpha}–/– mice (11). Because glucocorticoids are known to induce PPAR{alpha} activity (26), we asked the question whether PPAR{alpha} is involved in the diurnal variation of Elovl3 expression. To elucidate this further, we analyzed the Elovl3 mRNA expression in PPAR{alpha}-ablated mice. Livers from 8- to 10-wk-old male wild-type and PPAR{alpha}–/– mice were analyzed at different time points during the day (ZT0, ZT4, ZT10, ZT14). Surprisingly, no significant difference was observed between PPAR{alpha}–/– and wild-type animals (Fig. 6Go).


Figure 6
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  		FIG. 6. Diurnal Elovl3 expression in liver is PPAR{alpha} independent. Ten micrograms of RNA were analyzed in livers obtained from 8- to 10-wk-old male wild-type (wt) and PPAR{alpha}–/– mice at indicated time points around the day (ZT0, ZT4, ZT10, ZT14). Results shown are the means ± SEM of values from two animals at each time point.

 
Connection between Elovl3 expression and the peroxisomal ABC transporter ABCD2: cross talk between estrogen receptor and peroxisomes
In mammals, VLCFAs are selectively degraded by peroxisomal β-oxidation. The import of VLCFAs into peroxisomes is inferred to be via ATP-binding cassette transporters, the four members of the ABCD family (ABCD1 to ABCD4), by analogy with the work in yeast of Hettema et al. (37). Indeed, X-linked adrenoleukodystrophy, caused by a mutation in the gene encoding the peroxisomal transporter ABCD1 (38), is a disorder characterized by impaired peroxisomal β-oxidation and accumulation of saturated and monounsaturated VLCFAs in plasma and tissues (39). The VLCFA accumulated in X-linked adrenoleukodystrophy are found to be mostly of endogenous origin (40), thus pointing out a link to defective VLCFA synthesis by fatty acid elongases. A close homolog, the ABCD2 gene, also encoding an ABC peroxisomal transporter (41), is partially redundant with ABCD1 and can compensate for the loss of ABCD1 in terms of accumulation of C26:0 in the mouse (24), whereas mice lacking ABCD2 accumulate C26:0 in the neural tissue (23).

When analyzing the expression of Elovl1 and Elovl3 in different tissues such as liver and skin, in which Elovl3 normally is expressed, and different areas of the brain, from ABCD2 knockout mice, we found a significant increase in Elovl3 expression exclusively in liver, whereas Elovl1 expression was unchanged (Fig. 7AGo). In contrast, in the liver of transgenic mice with overexpression of the ABCD2 protein, Elovl3 expression was significantly reduced (Fig. 7BGo), whereas the Elovl1 expression was not affected, thus establishing a functional cross talk between Elovl3 and ABCD2. Although the level of expression was modified in the ABCD2 mutant mice, the diurnal variation was maintained (Fig. 7CGo).


Figure 7
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  		FIG. 7. Impaired Elovl3 expression in ABCD2 mutant mice. mRNA levels in liver and skin tissues obtained from wild-type (wt) and Abcd2–/– mice (A–) and analyzed by Northern blot (15 µg RNA) (A). Elovl1 and Elovl3 mRNA levels in liver (10 µg RNA) were analyzed in wild-type and transgenic mice overexpressing ABCD2 (A+) (B). Results shown are the means ± SEM of values from three animals at each time point. C, Elovl3 expression levels in liver from wt, A+, and Abcd2–/– mice at ZT0 and ZT12 analyzed by quantitative RT-PCR and normalized to 36b4 (also called Rpl0). Significant differences have been determined by ANOVA followed by Tukey honestly significant difference posttest [P ≤ 0.05 (*), P ≤ 0.01 (**), and P ≤ 0.001 (***)].

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Elovl3 is the only gene among the members of the Elovl family, of which expression has been shown to be radically altered under physiological conditions in mice. The gene was first identified as a cold inducible gene because the expression was highly elevated in brown adipose tissue upon cold exposure (10). The regulation of Elovl3 in brown adipose tissue has been further studied both in vivo and in primary cultures of brown adipocytes in which the expression has been shown to be under control of glucocorticoids, norepinephrine, and the three PPAR isoforms; PPAR{alpha}, PPAR{delta}, and PPAR{gamma} (10, 11, 12). Here we show that Elovl3 displayed a strong diurnal expression pattern exclusively in sexually mature male mouse liver, which in part could be explained by modulation of circulating glucocorticoid and androgen levels. In addition, in contrast to the expression of several enzymes involved in fatty acid synthesis, Elovl3 expression was not affected in liver by either fasting or refeeding. This implies that Elovl3 is not involved in insulin regulated fatty acid synthesis for storage under normal feeding conditions as most of the other lipogenic enzymes. Recent data rather suggest that the function of the ELOVL3 enzyme is to maintain the intracellular pool of specific VLCFAs to support the formation of lipid droplets within the cell, especially under conditions when the energy expenditure is increased (11, 16).

It is well known that daytime feeding for a period of time alters the daily glucocorticoid secretion in nocturnal animals independently of the light cycle (33). In this study, we show that there was a 12-h shift in Elovl3 expression in conditionally fed animals with the highest mRNA level at the beginning of the dark period, whereas Elovl1 expression was not particularly affected by restricted daytime feeding. Quite recently, in the course of this study, Anzulovich et al. (21) showed a circadian expression of Elovl3 in liver, which was abolished in Clock mutant mice. To synchronize physiology with external time, the suprachiasmatic nucleus emits signals via the hypophysical ACTH to a series of peripheral circadian clocks found in most organs. Among peripheral clocks, the one in the adrenal gland is particularly interesting because adrenal corticoids synchronize metabolic rhythms in liver (35). If the circadian expression of Elovl3 in liver is dependent on the levels of glucocorticoids, the expected latency period would be approximately 8 h. In animals injected ip with dexamethasone, there was a distinct increase in Elovl3 mRNA 8 h later, compared with control animals, suggesting that the elevated Elovl3 expression in liver, like in brown adipose tissue, is controlled by glucocorticoids. However, in contrast to liver, no sign of diurnal Elovl3 expression has been observed in either brown adipose tissue or the skin, which, except liver, are the tissues in which the highest Elovl3 expression has been detected.

In addition to fluctuating glucocorticoid levels within the circulation, 11β-hydroxysteroid dehydrogenase (11β-HSD1) constitutes a local determinant of the active glucocorticoid levels within peripheral tissues, as 11β-HSD1 catalyzes the interconversion of inactive keto- and active hydroxycorticosteroids in most tissues (42). The 11β-HSD1 mRNA level has been shown to be regulated in a similar way as Elovl3 in primary brown adipocytes (11). However, because we did not detect any clear circadian variation in the expression of 11β-Hsd1 in liver (Fig. 1AGo), we assumed that the glucocorticoid control of Elovl3 expression is dependent on the circadian release of glucocorticoids from the adrenal cortex. This is supported by data showing that the diurnal phase of circadian genes like Per, Cry, and Rev-erb{alpha} becomes entirely inverted locally in liver when feeding is restricted to daytime (31) or shifted by injection of dexamethasone into mice (35), which, taken together, suggest that diet intake directs Elovl3 expression via glucocorticoid signaling to the local clock in the liver.

Except glucocorticoids, our data also clearly revealed a role of gender-specific hormones in the control of Elovl3 expression in liver. Interestingly, and in strong contrast to Elovl3, Améen et al. (43) found higher mRNA levels of the lipogenic transcription factor SREBP-1c and FAS in females, whereas no sex difference was detected for ACC1 and SCD1 mRNA, which again underlines a different role in lipid synthesis for Elovl3 and the other lipogenic enzymes involved in fatty acid synthesis.

Several reports also underline the existence of a daily rhythm of androgens in both humans and rodents (44, 45). The oscillation of androstendione and testosterone secretion is, like for glucocorticoid secretion, controlled by ACTH release from the hypophysis showing a maximal secretion in late evening and minimal secretion in early morning in rodents. On the contrary, adult male rats display a diurnal variation for nuclear estrogen receptors in liver as well as serum levels of estradiol with the maximal level in the morning and the lowest level in the evening. Bryzgalova et al. (46) recently show that in liver of estrogen receptor-{alpha} (ER{alpha}) knockout female mice, Elovl3 expression is highly induced, which implies that estrogen suppresses Elovl3 expression under normal conditions. However, our analysis of ovariectomized mice suggests that reduced estrogen levels are not enough to stimulate Elovl3 expression in female mice. If estrogen receptor-{alpha} acts as a suppressor of Elovl3 expression in male mice, the lag phase of response is approximately 8 h, which is very similar to what was suggested for the positive glucocorticoid response in this study.

Our finding of sex difference in Elovl3 expression also raise the possibility that it is regulated by GH plasma level because gender-specific control of liver genes has been extensively associated with sexually dimorphic pattern of GH secretion (47). Accordingly, further studies are needed to explain the sex-dependent regulation of Elovl3 expression.

Nutritional studies on adult rats suggest that hepatic elongase expression might, in part, be controlled by PPAR{alpha} (30), which is generally accepted to be involved in activating genes coding for enzymes involved in catabolic processes like β-oxidation (48). Interestingly, here we show that the diurnal variation in liver of male mice was independent of PPAR{alpha}. However, our data on the ABCD2 mutant mice show that although the control of diurnal Elovl3 expression in liver is independent of PPAR{alpha}, it can be significantly elevated through a mechanism, which involves peroxisomal fatty acid oxidation. It has been shown that accumulation of VLCFAs leads to an up-regulation of β-oxidation enzymes in wild-type mice, but this mechanism is abolished in PPAR{alpha}–/– mice (49). However it is not known whether the impaired Elovl3 expression in the ABCD2 mutant mice is PPAR{alpha} dependent. Interestingly, although both Elovl3 and Elovl1 have been suggested to control the synthesis of saturated and monounsaturated VLCFAs, Elovl1 is ubiquitously expressed but not affected in ABCD2 mutant mice. Our results suggests that the levels of ABCD2 expression, and presumably of its transported substrates into or outside peroxisomes, might directly or indirectly modulate regulation of Elovl3 expression in mouse liver. Nevertheless, the precise molecular mechanisms linking peroxisomes with fatty acid elongation remains to be elucidated.

In conclusion, whereas the classical lipogenic enzymes such as FAS, ACC, and SCD1, which are highly expressed around ZT8-ZT22 in liver, are involved in fuel storage controlled by daily food intake, the circadian rhythm of Elovl3 was not altered by fasting and refeeding. Instead, our data here suggest that the diurnal variation of Elovl3 in liver is regulated and maintained by a coordinated release of different steroid hormones, i.e. glucocorticoids, androgens, and estrogens, from peripheral tissues, which, in turn, is defined by diurnal food intake in a more long-term perspective. However, to unravel the question why this is controlled differently in females and males, more detailed studies on endogenous lipid synthesis vs. food intake are needed in both liver and whole animals.


   Acknowledgments
 
We thank B. Leksell and Damir Zadravec for technical assistance and Dr. Elisabeth Metzger as mouse facility manager at the IGBMC (Strasbourg, France) for care of ABCD2 mice.


   Footnotes
 
This work was supported by grants from the Swedish Natural Science Research Council (to A.Jac.), the European Commission (LSHM-CT2004-502987), the European Leukodystrophy Association (ELA; 2006-043I4), and the Spanish Institute for Health Carlos III (FIS PI051118). The work was developed under the Cooperation of Science and Technology action BM0604 (to A.P.). S.F. was a fellow of the ELA (ELA 2007-018F4) and the European Commission.

First Published Online February 21, 2008

Abbreviations: ABCD, ATP-binding cassette, subfamily D(ALD); ACC, acetyl-CoA carboxylase; ELOVL, elongation of VLCFA; FAS, fatty acid synthase; 11β-HSD1, 11β-hydroxysteroid dehydrogenase; LXR, liver X receptor; PPAR, peroxisome proliferator-activated receptor; RT, reverse transcription; SCD1, sterol CoA desaturase-1; SDS, sodium dodecyl sulfate; SREBP, sterol regulatory element-binding protein; SSC, saline sodium citrate; VLCFA, very long chain fatty acid; ZT, Zeitgeber time.

Received October 12, 2007.

Accepted for publication February 14, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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