AJP - Endo Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 289: E517-E526, 2005. First published April 26, 2005; doi:10.1152/ajpendo.00045.2005
0193-1849/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/4/E517    most recent
00045.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jakobsson, A.
Right arrow Articles by Jacobsson, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jakobsson, A.
Right arrow Articles by Jacobsson, A.

Differential regulation of fatty acid elongation enzymes in brown adipocytes implies a unique role for Elovl3 during increased fatty acid oxidation

Andreas Jakobsson, Johanna A. Jörgensen, and Anders Jacobsson

The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden

Submitted 1 February 2005 ; accepted in final form 20 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The expression of the Elovl3 gene, which belongs to the Elovl gene family coding for microsomal enzymes involved in very long-chain fatty acid (VLCFA) elongation, is dramatically increased in mouse brown adipose tissue upon cold stimulation. In the present study, we show that the cold-induced Elovl3 expression is under the control of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) and that this regulation is part of a fundamental divergence in the regulation of expression for the different members of the Elovl gene family. In cultured brown adipocytes, a mixture of norepinephrine, dexamethasone, and the PPAR{alpha} ligand Wy-14643, which rendered the adipocytes a high oxidative state, was required for substantial induction of Elovl3 expression, whereas the same treatment suppressed Elovl1 mRNA levels. The nuclear liver X receptor (LXR) has been implicated in the control of fatty acid synthesis and subsequent lipogenic processes in several tissues. This regulation is also exerted in part by sterol regulatory element-binding protein (SREBP-1), which is a target gene of LXR. We found that stimulation of Elovl3 expression was independent of LXR and SREBP-1 activation. In addition, exposure to the LXR agonist TO-901317 increased nuclear abundance of LXR and mature SREBP-1 as well as expression of the elongases Lce and Elovl1 in a lipogenic fashion but repressed Elovl3 expression. A functional consequence of this was seen on the level of esterified saturated fatty acids, such as C22:0, which was coupled to Elovl3 expression. These data demonstrate differential transcriptional regulation and concomitantly different functional roles for fatty acid elongases in lipid metabolism of brown adipocytes, which reflects the metabolic status of the cells.

elongase; brown adipose tissue; gene expression; very long-chain fatty acid

FATTY ACIDS (FA) function not only as bulk molecules in membrane and storage lipids but have also been shown to exert signaling functions by modulation of nuclear receptor activation (4, 41). Such signaling processes implicate FA and related metabolites in cellular feedback functions to reflect the metabolic status of the cell.

The bulk of the FA synthesis in cells is performed by the cytosolic complex fatty acid synthase (FAS) (54). FAS produces medium- to long-chain FA (up to C16), which can be further elongated by membrane-bound enzymes mostly residing in the endoplasmatic reticulum (ER). Members of the Elovl (elongation of very long-chain fatty acids) gene family encode for enzymes, which are believed to perform the first step (condensation) in the elongation cycle to produce long-chain or very long-chain fatty acids (LCFA/VLCFA) from shorter FA. The family of enzymes consists of at least six members in mouse and human, believed to exert substrate-specific elongation with respect to FA length and unsaturation (30, 31, 36, 53, 61).

One important factor regulating FA metabolism in adipocytes is the transcription factor liver X receptor-{alpha} (LXR{alpha}) (26), which is activated by endogenous oxysterols. Along with the functionally overlapping LXR{beta} isoform, LXR{alpha} is involved in the control of cellular lipid homeostasis (24, 29) by activation of genes that are associated with FA and triglyceride synthesis, for example, FAS and sterol regulatory element-binding protein-1 (SREBP-1) (25, 44).

Two isoforms of SREBP-1 have been identified, SREBP-1a and SREBP-1c, originating from the same gene by the use of two different transcription start sites (60). Cleavage of the inactive SREBP precursor proteins residing in the ER, by a two-step proteolytic cascade, induces translocation of the NH2-terminal part into the nucleus, where it exerts its effects on transcription (21, 55, 59). One recently characterized member of the Elovl family, long-chain elongase (Lce), which has an apparent substrate specificity for saturated FA in the C12-C16 range, displays a lipogenic expression profile manifested in transgenic mice overexpressing constitutively active isoforms of SREBP-1 (36).

The Elovl1 and Elovl3 genes have been suggested to encode for proteins involved in the elongation of saturated and monounsaturated FA in the C20-C24 range. In similarity to Lce and Fas, Elovl1is ubiquitously expressed in mice, whereas Elovl3is expressed mainly in liver, skin, and brown adipose tissue (BAT) (53). In BAT, Elovl3 expression is very low when mice are housed at thermoneutrality but displays a dramatic upregulation during BAT activation and recruitment in response to cold stress (4°C for 3 days) (52). Increased VLCFA elongation activity in BAT in response to cold stress has previously been shown by Tvrdik et al. (53). BAT activation and recruitment processes, which include induction of Elovl3expression, are primarily controlled by sympathetic nerve activity and norepinephrine (NE) release. The resulting nonshivering thermogenesis primarily utilizes FA as substrate for mitochondrial respiration and subsequent heat generation through the uncoupling protein UCP1 (reviewed in Ref. 10).

Differentiation of isolated primary brown adipocytes in culture, indicated by multilocular fat accumulation and UCP1 expression, is spontaneously induced around day 5 after cell isolation, depending mainly on cell confluence and supply of nutrients from the culture medium. In cells that have already acquired adipocyte characteristics, NE advances final differentiation, including a dramatic increase in the expression of UCP1 and other genes associated with increased thermogenic capacity (reviewed in Refs. 10 and 39). However, to substantially induce Elovl3 expression in cultured cells, the addition of glucocorticoids together with chronic exposure to NE is required (52).

In the present study, we show that the induced Elovl3 expression in brown fat of cold-stimulated mice requires the transcription factor peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}). By using primary brown adipocyte cultures, we further demonstrate differential regulation between Elovl3 and the two other FA elongases, LceandElovl1, involved in the synthesis of saturated FA, which was correlated with FA oxidation processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. L-Norepinephrine bitartrate (NE; Arterenol), dexamethasone, TO-901317, 9-cis-retinoic acid, WY-14643, 22(S)-hydroxycholesterol, and all other chemicals when otherwise not stated, were obtained from Sigma. NE was dissolved in water, whereas all other compounds used for cell stimulation were dissolved in ethanol. The concentrations used for NE, dexamethasone, TO-901317, 9-cis-retinoic acid, and WY-14643 were chosen on the basis of titration experiments and shown to give the optimal estimated effects in primary cultured brown adipocytes.

Animals. PPAR{alpha}-ablated mice were a kind gift from Dr. F. Gonzales (28), backcrossed on a DBA background. As control mice, age-matched mice from this back-cross, which were bred under the same conditions as the PPAR{alpha}-ablated mice, was used. Animals were housed in groups of three to four mice per cage and were fed ad libitum (Rat and Mouse Standard Diet No. 1; BeeKay Feeds, B&K Universal, Stockholm, Sweden), had free access to water, and were kept on a 12:12-h light-dark cycle at room temperature. Before the cold exposure experiments, the mice were kept at 30°C (thermoneutrality) for 6 days. After this period, animals were exposed to 22°C for the indicated times or remained at 30°C.

Cell culture. Brown adipocyte precursors were isolated from the interscapular, axillary, and cervical BAT depots of 3- to 4-wk-old male mice of the NMRI strain (B&K) and the DBA strain containing a PPAR{alpha}-ablation (28) and grown in culture principally as described by Néchad et al. (37). Briefly, tissues were minced in HEPES-buffered solution containing 0.2% (wt/vol) collagenase type II (Sigma), and digestion was allowed for 30 min at 37°C. The digest was filtered through 250- and 25-µm nylon filters to remove undigested parts and mature cells. Precursor cells were pelleted by centrifugation (700 g), washed in Dulbecco's modified Eagle's medium, pelleted, and resuspended in 0.5 ml of culture medium/mouse. The cells were seeded in six-well plates (Corning) at a density of 1–2 x 104 cells/cm2. The cells were cultured in Dulbecco's modified Eagle's medium (1 g glucose/liter) supplemented with 10% newborn calf serum, 4 nM insulin, 25 µg/ml sodium ascorbate, 10 mM HEPES, 4 mM glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin at 37°C in an atmosphere of 8% CO2 in air. The cells were washed with Dulbecco's modified Eagle's medium on day 1, and the medium was changed on days 1, 3,and6 of culture. Administration of agents was performed as described in the figure legends, and the respective solvents (ethanol and water) were added to control wells to exclude unspecific effects.

RNA analysis. Cells were harvested in 1 ml of Ultraspec solution (Biotec, Houston, TX), and total RNA was isolated according to the procedure described by the manufacturer. Ten micrograms of total RNA were separated by electrophoresis in an ethidium bromide-containing agarose-formaldehyde gel and blotted onto Hybond N membranes (Amersham). Prehybridization and hybridization were carried out in 50% formamide, 5x SSC, 5x Denhardt's solution, 50 mM sodium phosphate, pH 6.5, 0.5% SDS, and 100 µg/ml degraded herring sperm DNA at 42°C overnight. Membranes were washed once 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 15–30 min. cDNA fragments, identified by BLAST (1) and verified by sequencing for Elovl1 and Elovl3 (53), SREBP-1 (51), 11{beta}-hydroxysteroid dehydrogenase 1 (11{beta}-HSD1; IMAGE EST clone gi:14605089), and LCE (gi:15715540), were [32P]dCTP-labeled using a random-primed DNA labeling kit (Amersham Biosciences) and used as probes. Phosphorimaging plates were exposed to the hybridized membranes for 24–72 h and subsequently analyzed in a FLA-3000 reader (Fuji). 18S rRNA was detected using a 473-nm laser in the FLA-3000 reader and used for normalization. Blots were quantified using Fuji Film Science Lab software and statistically analyzed by repeated-measures ANOVA and Tukey's multiple comparison test in GraphPad PRISM.

Isolation of subcellular protein fractions. Crude nuclear extracts were isolated according to Andrews and Faller (2). Medium was removed, and cells were scraped into 1 ml of cold phosphate-buffered saline, pelleted for 10 s, and resuspended in 400 µl of cold buffer A [10 mM HEPES-KOH, pH 7.9, at 4°C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, complete miniprotease inhibitor cocktail (Roche)] by flicking the tube. Cells were allowed to swell on ice for 10 min and were then vortexed for 10 s. Nuclei were pelleted by centrifugation at 2000 g for 1 min, and the supernatant (S1 fraction) was transferred to a new tube. Pellets were resuspended in 50 µl of cold buffer B [20 mM HEPES-KOH, pH 7.9, at 4°C, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, complete miniprotease inhibitor cocktail (Roche)] and incubated on ice for 20 min for high salt extraction. Cellular debris was removed by centrifugation (20.000 g) for 2 min at 4°C, and the supernatant (containing DNA-binding proteins) was stored at –80°C. The S1 fraction was centrifuged at 20,000 g for 15 min, and the supernatant was discarded, whereas the pellet (containing mitochondria and peroxisomes) was resuspended in 30 µl of cold buffer B and stored at –80°C. Protein concentration was determined by protein assay kit (Bio-Rad) for the isolated fractions before gel electrophoresis. Recovery of the nuclear and mitochondrial/peroxisomal proteins in the subcellular fractions was assessed by detection of marker proteins [cAMP response element-binding protein (CREB; not shown), UCP1, and acyl-CoA oxidase (AOx), respectively].

Protein analysis. Nuclear proteins (7.5 µg) and 3 µg of proteins from the 20,000-g fraction were separated on NuPAGE 10% Bis-Tris gels (Invitrogen) using NuPAGE MOPS-SDS running buffer according to the manufacturer's protocol and blotted onto nitrocellulose membranes. Membranes were blocked in 5% milk, washed in Tris-buffered saline containing 0.1% Tween-20 (TBST), incubated with primary antibody diluted in TBST and 5% BSA overnight, washed three times in TBST, and incubated with horseradish peroxidase-linked secondary antibody (Cell Signaling) for 1 h. Proteins were visualized using the ECLplus kit (Amersham Biosciences) and detected in a LAS-1000 charge-coupled device camera (Fuji). Proteins were quantified and statistically analyzed as described for RNA. SREBP-1 antibodies were purchased from Santa Cruz Biotechnologies and diluted 1:500, UCP1 antibodies were generated by Neosystem (COOH-terminal part, 1:2,000 dilution), and AOx (58) and multifunctional enzyme type 1 (MFE-1) (42) antibodies (1:2,000 dilution) were kindly provided as a gift from Dr. S. H. Alexsson, Karolinska Hospital and Dr. J. K. Hiltunen, University of Oulu.

Lipid analysis. Exogenous [14C]palmitic acid (0.1 µCi/ml, 16 µM; GE Healthcare) bound to BSA (0.5%) was added to the culture medium 4 h before harvest of the cells. Cells were scraped in cold phosphate-buffered saline, pelleted, vigorously vortexed in the lipid extraction solvent mixture chloroform(C)-methanol(M)-water (4:8:3) for 1 min, left at room temperature for 20 min, and reextracted in C-M (2:1). Extracts were pooled, evaporated in speed-vac and redissolved in C-M (2:1). Lipid aliquots were applied to a silica gel 60 TLC plate (VWR International), and the plate was developed as previously described by Downing (13). Briefly, the plate was prewashed with chloroform and placed in 105°C for 30 min before sample application. Development was as follows: first phase using hexane (to the end of the plate), air dried for 15 min; second phase using toluene (end of the plate), air dried for 15 min; third phase containing hexane-ether-acetic acid (70:30:1) run on half the plate. The thin-layer chromatography (TLC) plate was sprayed with sulfuric acid-ethanol (1:1) and charred at 150°C. Lipids were detected in the LAS-1000 and quantified as described earlier. Lipid identities were determined by standards run in parallel.

FA analysis. Cellular lipids were extracted in the presence of authentic internal standards by the method of Folch et al. (14) using chloroform-methanol (2:1, vol/vol). Approximately 25 million cells per treatment from three independent experiments were pooled and used for FA analysis. Individual lipid classes within each extract were separated by preparative TLC, as described previously (56). Authentic lipid class standard compounds were spotted onto the two outside lanes of the TLC plate to enable localization of the sample lipid classes. Each lipid fraction was scraped from the plate and trans-esterified in 3 N methanolic-HCl in a sealed vial under a nitrogen atmosphere at 100°C for 45 min. The resulting FA methyl esters were extracted from the mixture with hexane containing 0.05% butylated hydroxytoluene and prepared for gas chromatography by sealing the hexane extracts under nitrogen.

FA methyl esters were separated and quantified by capillary gas chromatography using a gas chromatograph (Hewlett-Packard model 6890, Wilmington, DE) equipped with a 30-m DB-225MS capillary column (J&W Scientific, Folsom, CA) and a flame ionization detector as described previously (56).

FA oxidation. Fifty microliters of NCSII tissue solubilizer (GE Healthcare) was applied on pieces of filter paper left to dry overnight. [14C]palmitic acid (0.1 µCi/ml, 16 µM; GE Healthcare) was added to cell culture wells as described for lipid analysis, and the culture plates were sealed using super sealing film (Life systems) with NCSII-treated filter papers attached over each well to collect released CO2 from cells. After 4 h of incubation at 37°C, filters were collected and the amount absorbed CO2 was quantified using liquid scintillation counting.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Reduced Elovl3 expression in BAT of cold-exposed PPAR{alpha}-ablated mice. In liver, PPAR{alpha} controls the expression of several genes involved in FA oxidation (3). Because the adrenergic pathway stimulates UCP1-mediated heat production, via increased FA {beta}-oxidation and uncoupled cellular respiration (reviewed in Ref. 10), along with increased Elovl3 synthesis (52), we investigated whether Elovl3 expression was effected in PPAR{alpha}-ablated mice upon cold stimulation. As seen in Fig. 1A, the expression of Elovl3 was severely reduced in the PPAR{alpha}-ablated mice after both 12 h and 3 days of mild cold exposure. This effect was specific for Elovl3, since the mRNA levels for the elongase Elovl1 was not affected by the PPAR{alpha} ablation.



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 1. Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha})-regulated Elovl3 mRNA expression in conjunction with increased fatty acid (FA) oxidation in brown adipocytes.A: representative Northern blots showing RNA levels of Elovl3, Elovl1, and ribosomal RNA, respectively, in control and PPAR{alpha}-ablated mice exposed to 30 or 22°C for indicated times. RNA levels were quantified, indicated as fold induction, by densitometric analysis. Primary brown adipocytes were cultured for 6 days up to confluence. From day 6, cells were treated (when indicated) with 0.1 µM norepinephrine (N) (every 12 h) and/or 1 µM dexamethsone (D) for 3 days. Wy-14643 (20 µM, W) and/or 500 nM 9-cis-retinoic acid (R) was added (when indicated) on day 7.5 (for 36 h). B: proteins in subcellular fraction containing mitochondria and peroxisomes were separated on SDS-PAGE, and typical marker proteins of FA {beta}-oxidation, acyl-CoA oxidase 1 (AOx), multifunctional enzyme type 1 (MFE-1), and uncoupling protein-1 (UCP1), were detected by immunoblotting and quantified. C: oxidation of palmitic acid was measured on day 9 as released CO2. Bars represent means ± SE of 4 experiments performed in triplicate. The difference between the 2 groups was not statistically significant. D: total RNA was isolated on day 9 and analyzed by Northern blot. Membranes were probed with Elovl3 cDNA and results normalized to 18S rRNA (see EXPERIMENTAL PROCEDURES). Bars represent means ± SE of 3 experiments performed in duplicate (autoradiogram shows representative blot). Significance at **P < 0.01 and ***P < 0.001 by comparing Elovl3 mRNA levels in respective treatments with (NDRW; set to 100%). E: total RNA isolated from NDRW-stimulated cells, as in D, obtained from control and PPAR{alpha}-ablated mice.

 
Elevated Elovl3 expression and FA oxidation by PPAR{alpha} ligand Wy-14643 in brown fat primary cultures. Cultured primary brown adipocytes are the only cell culture system described that expresses significant amounts of endogenous Elovl3. As shown earlier, 3 days of chronic NE stimulation in combination with glucocorticoids (dexamethasone) induces the expression of Elovl3 in cultured brown adipocytes (52). Because NE, in conjunction with PPAR{alpha}, stimulates the expression of Elovl3 in vivo as shown above, we asked whether Elovl3 expression could be augmented along with improved thermogenic capacity of the cultured brown adipocytes by stimulating the cells with a synthetic PPAR{alpha} agonist (Wy-14643). Activation of PPAR{alpha} has been shown to increase the rate of {beta}-oxidation through upregulation of both mitochondrial and peroxisomal enzymes (6, 17, 27). As seen in Fig. 1B, Wy-14643, both alone (W) and in combination with NE, dexamethasone, and RXR ligand 9-cis-retinoic acid (heterodimerizing partner of PPARs) (NDRW), increased the level of the peroxisomal enzymes AOx and MFE-1, which are known PPAR{alpha} target genes. Wy-14643 also induced an increase in the mitochondrial protein content of UCP1 when added in combination with adrenergic, retinoid, and glucocorticoid stimuli (NDRW), which implies an increased capacity for uncoupled respiration. In agreement with this, the oxidative response to adrenergic stimuli and PPAR{alpha} activation (NDRW), measured by released CO2 generated from {beta}-oxidation of exogenously added labeled palmitic acid, was also increased compared with vehicle-treated cells (Fig. 1C). The level of Elovl3expression correlated with the oxidative capacity of the brown adipocytes; i.e., it was further elevated by the addition of PPAR{alpha} ligand Wy-14643 (Fig. 1D). In accord with the in vivo data, the Elovl3 expression was markedly reduced in NDRW-stimulated brown adipocytes obtained from PPAR{alpha}-ablated mice (Fig. 1E). As expected, no expression was detected by Northern blot in unstimulated cells. Elovl3 expression could not be detected in wild-type cells stimulated with dexamethasone, WY-14643, or 9-cis-retinoic acid alone (not shown), implying that adrenergic stimuli are essential for the activation processes involving elevated Elovl3 expression in brown adipocytes.

Induction of Elovl3 expression is not mediated via induction of lipogenic factors LXR and SREBP-1. Activation of LXRs by natural and synthetic agonists induces SREBP-1 mRNA and protein expression, and subsequently also elevated levels of mature SREBP-1 in the nucleus, which in turn can induce transcription of downstream lipogenic genes as shown for Fas and Lce (7, 36). To elucidate the signals inducing Elovl3 expression, we investigated whether the NDRW mixture activated the transcription factors LXR and SREBP-1 in brown adipocytes. Interestingly, no increase in the nuclear abundance of SREBP-1 was detected when maximal induction of Elovl3 expression was obtained (Fig. 2, A and B).



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 2. Effects of nuclear receptor agonists and adrenergic brown adipocyte activation on abundance of sterol regulatory element-binding protein (SREBP)-1. Brown adipocytes were cultured and treated as described in Fig. 1. SREBP-1 mRNA levels (A) and SREBP-1 nuclear protein levels (B) were analyzed following indicated treatments. Visible bands are 1a and 1c isoforms, as indicated by arrows. TO-901317 (T1317) and 22(S)-hydroxycholesterol (22(S)-HC) (20 µM) were added to cells on day 7.5 (for 36 h). Bars represent 3 individual experiments in duplicate ± SE. Significance at *P < 0.05, **P < 0.01, and ***P < 0.001.

 
To further investigate whether LXR- and SREBP-1-mediated signaling pathways can, in fact, be activated in cultured primary brown adipocytes and, as a consequence, stimulate Elovl3 expression, cells were stimulated with the synthetic LXR agonist TO-901317. Ligand activation increased the amount of SREBP-1 mRNA and nuclear protein levels (Fig. 2, A and B). In contrast, NDRW-induced Elovl3 mRNA levels were markedly suppressed by the LXR agonist (Fig. 3A). In addition, Elovl3 expression was not restored upon simultaneous addition of 22(S)-hydroxycholesterol, which inhibits SREBP-1 cleavage and nuclear translocation without interfering with activation of LXR (11, 23, 55) (Figs. 2B and 3A). This suggests that changes in nuclear levels of SREBP-1 did not contribute to the LXR-mediated suppression of Elovl3 mRNA levels in NDRW-stimulated brown adipocytes. In contrast, and as expected, the expression profile of the two other elongases, Elovl1 and Lce, followed the level of nuclear SREBP-1 (Figs. 2B and 3, B and C).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3. Contrasted Elovl3 mRNA expression pattern from Elovl1 and Lce. Cells were cultured and treated as described in Fig. 1. Shown are relative mRNA levels following indicated treatments, as in Fig. 2, for Elovl3(A), Elovl1(B), and Lce(C), analyzed by Northern blot. TO-901317 treatment is set to 100%, except for Elovl3where NDRW represents 100%. Bars represent 3 individual experiments in duplicate ± SE. Significance at *P < 0.05, **P < 0.01, and ***P < 0.001.

 
Elovl3 expression correlated with expression of the main regulatory enzyme in glucocorticoid metabolism. Because the regulation of Elovl3 expression did not follow an ordinary lipogenic pattern controlled by LXR and SREBP-1, we asked whether other factors related to lipid metabolism would show a similar expression profile to Elovl3. We (52) have previously shown glucocorticoid stimulation of Elovl3 gene expression in cultured brown adipocytes, which is also shown here in Fig. 1. The glucocorticoid-metabolizing enzyme 11{beta}-HSD1 primarily activates cellular glucocorticoids to sustain proper glucocorticoid signaling, although the enzyme has also been suggested to catalyze the reverse, inactivating reaction at a slower rate. To investigate whether Elovl3 expression was paralleled by changes in the regulation of glucocorticoid turnover, 11{beta}-HSD1 expression was measured. As shown for Elovl3, a combination of NE and dexamethasone (NDRW mixture) increased the11{beta}-HSD1 expression, whereas LXR activation decreased11{beta}-HSD1 mRNA levels (Fig. 4) in parallel with Elovl3expression. Again, similar to Elovl3 expression, the TO-901317-induced suppression was not blocked by addition of 22(S)-hydroxycholesterol, why a SREBP-1 dependent mechanism was excluded. This suggests that the regulation of local glucocorticoid activation and Elovl3-expression in BAT are closely associated and in a similar way restrained by LXR.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 4. Liver X receptor (LXR) agonist suppresses 11{beta}-hydroxysteroid dehydrogenase-1 (11{beta}-HSD1) mRNA levels. 11{beta}-HSD1 mRNA levels in brown adipocytes analyzed by Northern blot, following indicated treatments as in Fig. 2. Bars represent 3 individual experiments in duplicate ± SE. Significance at *P < 0.05, **P < 0.01, and ***P < 0.001.

 
Elovl3 expression and lipid composition. To assess a functional consequence of altered Elovl3 expression in the brown adipocytes, we analyzed the lipid composition by TLC and gas chromatography as described in MATERIALS AND METHODS. In NDRW-treated cells where Elovl3 expression was high, incorporation of 14C label (originating from exogenous FA) into sterol esters and fatty alcohols was selectively increased (Fig. 5A). This effect was abolished by the addition of LXR agonist, both in the presence and in the absence of 22(S)-hydroxycholesterol, as shown earlier to inhibit SREBP maturation. In addition, in NDRW-treated cells, 22(S)-hydroxycholesterol did not have any effect on the incorporation of 14C label into sterol esters and fatty alcohols, which was in accord with sustained high Elovl3 expression in these cells.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 5. Elovl3 expression and FA incorporation. A: thin-layer chromatography (TLC) separation of labeled cellular lipids from total lipid extract. Incorporation of 14C label originating from [U-14C]palmitic acid into cellular lipids was detected by phosphorimaging after TLC separation and identified by lipid standards run in parallel. Cells were cultured and treated as described in Fig. 2. [U-14C]palmitic acid was added 4 h before harvest. Samples are shown in triplicates. B: content of palmitic (16:0) and behenic acid (C22:0) expressed as normalized mole percent of total FA content within the sterol ester fraction, analyzed by gas chromatography. C: scheme FA esterification pathways. MG, monoglycerides; DG, diglycerides; TG, triglycerides; SE, sterol esters; Falc, fatty alcohols.

 
To look for changes in specific FA incorporation, which could be linked to the level of Elovl3expression, we analyzed the sterol ester fractions further. To obtain a sufficient amount of material for measurements of the FA composition, ~25 million cells per treatment were pooled from three independent experiments. Noticeably, the FA composition in the sterol ester fractions displayed alterations in behenic acid (22:0) content between the different treatments (Fig. 5B), which was in accordance with the amount of incorporated FA label into sterol esters (Fig. 5A) and the level of Elovl3 expression (Fig. 3A); high levels in NDRW-treated cells and low levels in cells stimulated by the LXR agonist. The alterations in sterol ester behenic acid content was specific, as no similar changes was seen for LCFA, such as palmitic acid (16:0; Fig. 5B), in response to the respective treatments. This is of particular interest because Elovl3 is suggested to be involved in the synthesis of saturated VLCFA in that range of C22–24 acyl-chain lengths (Ref. 53 and A. Jacobsson, unpublished observations).

These data suggest a function for Elovl3 in the formation of saturated VLCFA, such as behenic acid, in a situation when the rate of cellular FA oxidation is stimulated. Ligand activation of LXR appears to inhibit similar lipid synthesis, in conjunction with a suppressive effect on Elovl3 expression.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The stimulatory effect of LXR agonists on genes involved in FA synthesis has been suggested to be mediated both directly through LXR and via SREBP-1c (25, 45). From our experiments, we conclude that, although LXR and SREBP-1 could be activated in primary brown adipocytes in response to a synthetic LXR agonist, the induction of Elovl3 expression by NDRW stimuli was not mediated via activation of LXR- or SREBP-mediated signaling pathways. In contrast to Lce and Elovl1, the LXR agonist TO-901317 exerted a negative effect on Elovl3 expression, although all of these three Elovl enzymes have been suggested to elongate saturated FA. Stimulation of Elovl3 expression was instead accomplished by the addition of a stimulatory mix including a PPAR{alpha} agonist, which proved to be a unique regulatory feature among the Elovl genes in the present study. The PPAR{alpha} regulation of Elovl3 expression in brown adipocytes was confirmed both in cultured cells obtained from PPAR{alpha}-ablated mice and in whole animals when exposed to cold.

PPAR{alpha} and LXR{alpha} have been proposed to form atypical heterodimers, working as negative regulators of the respective target genes (15, 35). The suppressive effect of LXR agonist on PPAR{alpha} agonist-stimulated Elovl3expression putatively suggests similar regulation in BAT, but the same relative suppression of Elovl3 expression in response to LXR agonist was seen when 9-cis-retinoic acid and Wy-14643 was excluded from the stimulatory mix. This indicates that the suppressive effect on Elovl3 levels is not dependent on downregulation of PPAR{alpha}- and RXR-mediated signals.

The assembly of nuclear receptor coactivator/corepressor complexes is an important determinant of the impact on transcriptional activity. Recent data on transcriptional control of lipid synthesis by the PPAR{gamma} coactivators PGC-1{alpha} and PGC-1{beta}, which have been shown to coactivate, e.g., PPAR{alpha} and LXR{alpha}, suggest a gene-specific displacement of corepressors by PGC-1{alpha} upon nuclear receptor ligand activation (16) and PGC-1{beta} mediated activation during energy storage (33). A specific recruitment of PGC-1{alpha} and PGC-1{beta} on different promoters, such as for Lce, Elovl1, and Elovl3, may explain, in part, the discrepancy in regulation by LXR on the expression of these genes.

SREBPs are important transcription factors in the regulation of lipogenic processes, as many of the target genes are involved in the synthesis of lipids. SREBP-2 is suggested to primarily regulate cholesterol homeostasis, whereas the two characterized isoforms of SREBP-1 (a and c) are implicated in FA and triglyceride synthesis, as suggested from studies in transgenic mice (19, 47, 48). Although several studies indicate distinct functions for the two SREBP-1 isoforms (20, 32, 49), we could not detect any difference in the induction or translocation of the mature SREBP-1a and -1c proteins in response to LXR agonist (Fig. 3C).

A possible candidate to be involved in the regulation of Elovl3 expression is the main regulatory enzyme in glucocorticoid metabolism, 11{beta}-HSD1, which proved to be regulated in a similar way to Elovl3 at the mRNA level in primary brown adipocytes. In white adipose tissue, glucocorticoids enhance adipogenesis by inducing 11{beta}-HSD1 activity in preadipocytes, which creates a feed-forward loop contributing to adipocyte differentiation (9, 18). In addition to fluctuating glucocorticoid levels in the circulation, this enzyme constitutes a local determinant of the active glucocorticoid levels within peripheral tissues, as 11{beta}-HSD1 catalyzes the interconvertion of inactive keto- and active hydroxycorticosteroids in most tissues favoring the dehydrogenase (activating) reaction. In this context, it is noteworthy that the synthetic glucocorticoid dexamethasone is also metabolized by the normal cellular mechanisms, but at a slower rate compared with endogenous glucocorticoids due to its structural properties (12). A decrease in the cellular expression of 11{beta}-HSD1 in response to LXR activation, which has previously also been shown in 3T3-L1 cells by Stulnig et al. (50), may result in lower levels of endogenous as well as synthetic active glucocorticoids. Although the enzyme may play a more-or-less different role in BAT than in white adipose tissue, the inhibitory effect of TO-901317 on Elovl3 expression may in part be explained by a decrease in glucocorticoid receptor signaling (Fig. 6).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 6. Regulation of Elovl genes in brown adipocytes. Schema of hormonal and metabolic regulation of Elovl genes with respect to regulatory factors correlated with oxidative or lipogenic events in brown adipocytes. nSREBP-1, SREBP-1; GR, glucocorticoid receptor; GCi, inactive glucocorticoid; GCa, active glucocorticoid; AR, adrenoreceptors; NE, norepinephrine.

 
In contrast to our findings, Hummasti et al. (22) recently reported a positive effect of LXR agonist GW-3965 on Elovl3 expression in differentiated 3T3-L1 white adipocytes. However, gene expression in a highly oxidative tissue such as BAT and a lipid storage tissue such as white adipose tissue may be subject to differential regulation as a consequence of different sets of transcription factors and their cell-specific modulations. In addition, Elovl3 expression is normally very low in white adipose tissue, and the ways of inducing adipocyte differentiation in cultured cell lines, such as 3T3-L1, may interfere with the physiological response to various stimuli. Analysis of the Elovl3 promoter region revealed putative binding sites for, e.g., cAMP response element-binding protein (CREB; NE-cAMP stimulation), PPAR{alpha}, and glucocorticoid receptor (GR). Whether any of these elements is actually involved in the Elovl3 expression has yet to be investigated.

The physiological activator of brown adipocytes, NE, confers the basis for induction of FA oxidation, including peroxisomal {beta}-oxidation (38), and thermogenesis in BAT.

The stimuli required for induction of Elovl3 expression, further emphasizes the "unique" cellular condition leading to transcription of this gene. As Elovl3 mRNA levels are correlated with increased {beta}-oxidation, the function of Elovl3 may be to replenish intracellular pools of specific FAs when the FA turnover rate is high. In vitro elongation experiments on microsomes from murine BAT, as well as functional complementation studies in yeast, indicated ELOVL3 activity for saturated and monounsaturated FA substrates in the C16-C24 range (53) (A. Jacobsson, unpublished observations). Our data from this study on the correlation between cellular behenic acid (22:0) content and the level of Elovl3 expression are in accordance with this. Even though we did not see any major differences between the different treatments in total FA incorporation within the triglyceride fraction, we cannot rule out that there is a specific difference in FA content similar to the sterol ester factions. However, the analysis was not readily performed due to very high basal FA incorporation.

Although the FA composition in both phospholipids and triglycerides from BAT is altered toward increased content of saturated C22 and C24 species in response to cold acclimation (40, 46), which is in agreement with elevated VLCFA elongation activity in cold-acclimated mice (53), the rationale for a shift toward saturated VLCFA in the cold is not clear. An increased amount of saturated VLCFA within the phospholipids would result in a more ordered (rigid) membrane structure to maintain barrier integrity during thermogenesis. It has also been shown that long hydrophobic FAs are important components in the formation of fat droplets (43). Although there is a decrease in triglyceride content during cold exposure, there is at the same time an increased triacylglycerol/FA turnover in BAT, which can be detected already during the first day of cold exposure (8, 34). The decrease in triacylglycerol content seen with NE-mediated BAT activation is secondary to activation of FA oxidation (5). Our data on Elovl mRNA levels support a role for ELOVL3 during intense FA turnover. Recent data show an impaired sterol ester and triglyceride formation in the sebocytes of Elovl3-ablated mice (57), suggesting that the function for ELOVL3 in brown adipocytes relates to the need for restoration of specific intracellular lipid pools when the FA turnover rate is high.

In conclusion, functional diversity and specificity of FA-modifying enzymes enables the cell to maintain a dynamic metabolic balance in response to hormonal signals and varying nutritional status. Our data show a fundamental divergence in transcriptional regulation of the genes controlling saturated FA synthesis in the cell, which implicates certain functions of these elongases in different metabolic contexts.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Swedish Research Council (to A. Jacobsson)


   ACKNOWLEDGMENTS
 
We thank B. Leksell and Dr. R. Westerberg for excellent technical assistance; Dr. J. K. Hiltunen for the kind gift of MFE-1 antibody; Dr. F. Gonzalez, National Cancer Institute, for kindly providing the PPAR{alpha}-ablated mice; Dr. S. Alexsson for kindly providing antibodies and valuable discussion; and Drs. J. Lindquist and J. Nedergaard for valuable discussion and manuscript revision.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Jacobsson, The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-10691 Stockholm, Sweden (e-mail: anders.jacobsson{at}wgi.su.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Altschul SF, Gish W, Miller W, Myers EW, and Lipman DJ. Basic local alignment search tool. J Mol Biol 215: 403–10, 1990.[CrossRef][Web of Science][Medline]
  2. Andrews N and Faller D. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 19: 2499, 1991.[Free Full Text]
  3. Aoyama T, Peters JM, Iritani N, Nakajima T, Furihata K, Hashimoto T, and Gonzalez FJ. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha). J Biol Chem 273: 23–28, 1998.[Abstract/Free Full Text]
  4. Auwerx J. Regulation of gene expression by fatty acids and fibric acid derivatives: an integrative role for peroxisome proliferator activated receptors. Horm Res 38: 269–277, 1992.[Web of Science][Medline]
  5. Baht HS and Saggerson ED. Effect of noradrenaline on triacylglycerol synthesis in rat brown adipocytes. Biochem J 258: 369–373, 1989.[Web of Science][Medline]
  6. Barbera MJ, Schluter A, Pedraza N, Iglesias R, Villarroya F, and Giralt M. Peroxisome proliferator-activated receptor alpha activates transcription of the brown fat uncoupling protein-1 gene. A link between regulation of the thermogenic and lipid oxidation pathways in the brown fat cell. J Biol Chem 276: 1486–1493, 2001.[Abstract/Free Full Text]
  7. Bennett MK, Lopez JM, Sanchez HB, and Osborne TF. Sterol regulation of fatty acid synthase promoter. Coordinate feedback regulation of two major lipid pathways. J Biol Chem 270: 25578–25583, 1995.[Abstract/Free Full Text]
  8. Brooks BJ, Arch JR, and Newsholme EA. Effect of some hormones on the rate of the triacylglycerol/fatty-acid substrate cycle in adipose tissue of the mouse in vivo. Biosci Rep 3: 263–267, 1983.[CrossRef][Web of Science][Medline]
  9. Bujalska IJ, Kumar S, Hewison M, and Stewart PM. Differentiation of adipose stromal cells: the roles of glucocorticoids and 11beta-hydroxysteroid dehydrogenase. Endocrinology 140: 3188–3196, 1999.[Abstract/Free Full Text]
  10. Cannon B and Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev 84: 277–359, 2004.[Abstract/Free Full Text]
  11. Cases S, Novak S, Zheng YW, Myers HM, Lear SR, Sande E, Welch CB, Lusis AJ, Spencer TA, Krause BR, Erickson SK, and Farese RVJ. ACAT-2, a second mammalian acyl-CoA:cholesterol acyltransferase. Its cloning, expression, and characterization. J Biol Chem 273: 26755–26764, 1998.[Abstract/Free Full Text]
  12. Diederich S, Eigendorff E, Burkhardt P, Quinkler M, Bumke-Vogt C, Rochel M, Seidelmann D, Esperling P, Oelkers W, and Bahr V. 11Beta-hydroxysteroid dehydrogenase types 1 and 2: an important pharmacokinetic determinant for the activity of synthetic mineralo- and glucocorticoids. J Clin Endocrinol Metab 87: 5695–5701, 2002.[Abstract/Free Full Text]
  13. Downing DT. Photodensitometry in the thin-layer chromatographic analysis of neutral lipids. J Chromatogr 38: 91–99, 1968.[CrossRef][Web of Science][Medline]
  14. Folch J, Lees M, and Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226: 497–509, 1957.[Free Full Text]
  15. Gbaguidi GF and Agellon LB. The atypical interaction of peroxisome proliferator-activated receptor alpha with liver X receptor alpha antagonizes the stimulatory effect of their respective ligands on the murine cholesterol 7alpha-hydroxylase gene promoter. Biochim Biophys Acta 1583: 229–236, 2002.[Medline]
  16. Guan HP, Ishizuka T, Chui PC, Lehrke M, and Lazar MA. Corepressors selectively control the transcriptional activity of PPARgamma in adipocytes. Genes Dev 19: 453–461, 2005.[Abstract/Free Full Text]
  17. Gulick T, Cresci S, Caira T, Moore DD, and Kelly DP. The peroxisome proliferator-activated receptor regulates mitochondrial fatty acid oxidative enzyme gene expression. Proc Natl Acad Sci USA 91: 11012–11016, 1994.[Abstract/Free Full Text]
  18. Hauner H, Schmid P, and Pfeiffer EF. Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells. J Clin Endocrinol Metab 64: 832–835, 1987.[Abstract/Free Full Text]
  19. Horton JD, Shimomura I, Brown MS, Hammer RE, Goldstein JL, and Shimano H. Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2. J Clin Invest 101: 2331–2339, 1998.[Web of Science][Medline]
  20. Horton JD, Shimomura I, Ikemoto S, Bashmakov Y, and Hammer RE. Overexpression of sterol regulatory element-binding protein-1a in mouse adipose tissue produces adipocyte hypertrophy, increased fatty acid secretion, and fatty liver. J Biol Chem 278: 36652–36660, 2003.[Abstract/Free Full Text]
  21. Hua X, Nohturfft A, Goldstein JL, and Brown MS. Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein. Cell 87: 415–426, 1996.[CrossRef][Web of Science][Medline]
  22. Hummasti S, Laffitte BA, Watson MA, Galardi C, Chao LC, Ramamurthy L, Moore JT, and Tontonoz P. Liver X receptors are regulators of adipocyte gene expression but not differentiation: identification of apoD as a direct target. J Lipid Res 45: 616–625, 2004.[Abstract/Free Full Text]
  23. Janowski BA, Grogan MJ, Jones SA, Wisely GB, Kliewer SA, Corey EJ, and Mangelsdorf DJ. Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta. Proc Natl Acad Sci USA 96: 266–271, 1999.[Abstract/Free Full Text]
  24. Janowski BA, Willy PJ, Devi TR, Falck JR, and Mangelsdorf DJ. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature 383: 728–731, 1996.[CrossRef][Medline]
  25. Joseph SB, Laffitte BA, Patel PH, Watson MA, Matsukuma KE, Walczak R, Collins JL, Osborne TF, and Tontonoz P. Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors. J Biol Chem 277: 11019–11025, 2002.[Abstract/Free Full Text]
  26. Juvet LK, Andresen SM, Schuster GU, Dalen KT, Tobin KA, Hollung K, Haugen F, Jacinto S, Ulven SM, Bamberg K, Gustafsson JA, and Nebb HI. On the role of liver X receptors in lipid accumulation in adipocytes. Mol Endocrinol 17: 172–182, 2003.[Abstract/Free Full Text]
  27. Keller H, Dreyer C, Medin J, Mahfoudi A, Ozato K, and Wahli W. Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers. Proc Natl Acad Sci USA 90: 2160–2164, 1993.[Abstract/Free Full Text]
  28. Lee SST, Pineau T, Drago J, Lee EJ, Owens JW, Kroetz DL, Fernandez-Salguero PM, Westphal H, and Gonzalez FJ. Targeted disruption of the {alpha} isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol 15: 3012–3022, 1995.[Abstract]
  29. Lehmann JM, Kliewer SA, Moore LB, Smith-Oliver TA, Oliver BB, Su JL, Sundseth SS, Winegar DA, Blanchard DE, Spencer TA, and Willson TM. Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway. J Biol Chem 272: 3137–3140, 1997.[Abstract/Free Full Text]
  30. Leonard AE, Bobik EG, Dorado J, Kroeger PE, Chuang LT, Thurmond JM, Parker-Barnes JM, Das T, Huang YS, and Mukerji P. Cloning of a human cDNA encoding a novel enzyme involved in the elongation of long-chain polyunsaturated fatty acids. Biochem J 350: 765–770, 2000.
  31. Leonard AE, Kelder B, Bobik EG, Chuang LT, Lewis CJ, Kopchick JJ, Mukerji P, and Huang YS. Identification and expression of mammalian long-chain PUFA elongation enzymes. Lipids 37: 733–740, 2002.[Web of Science][Medline]
  32. Liang G, Yang J, Horton JD, Hammer RE, Goldstein JL, and Brown MS. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem 277: 9520–9528, 2002.[Abstract/Free Full Text]
  33. Lin J, Yang R, Tarr PT, Wu PH, Handschin C, Li S, Yang W, Pei L, Uldry M, Tontonoz P, Newgard CB, and Spiegelman BM. Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP. Cell 120: 261–273, 2005.[CrossRef][Web of Science][Medline]
  34. Lindberg O, Bieber LL, and Houstek J. Brown adipose tissue metabolism: an attempt to apply results from in vitro experiments on tissue in vivo. In: Regulation of Depressed Metabolism and Thermogenesis, edited by Jansky L and Musacchia XJ. Xxxx, OH: Thomas Springfield, 1976, p. 117–136.
  35. Miyata KS, McCaw SE, Patel HV, Rachubinski RA, and Capone JP. The orphan nuclear hormone receptor LXR alpha interacts with the peroxisome proliferator-activated receptor and inhibits peroxisome proliferator signaling. J Biol Chem 271: 9189–9192, 1996.[Abstract/Free Full Text]
  36. Moon YA, Shah NA, Mohapatra S, Warrington JA, and Horton JD. Identification of a mammalian long chain fatty acyl elongase regulated by sterol regulatory element-binding proteins. J Biol Chem 276: 45358–45366, 2001.[Abstract/Free Full Text]
  37. Néchad M, Kuusela P, Carneheim C, Björntorp P, Nedergaard J, and Cannon B. Development of brown fat cells in monolayer culture. I. Morphological and biochemical distinction from white fat cells in culture. Exp Cell Res 149: 105–118, 1983.[CrossRef][Web of Science][Medline]
  38. Nedergaard J, Alexson S, and Cannon B. Cold adaptation in the rat: increased brown fat peroxisomal {beta}-oxidation relative to maximal mitochondrial oxidative capacity. Am J Physiol Cell Physiol 239: C208–C216, 1980.[Abstract/Free Full Text]
  39. Nedergaard J, Herron D, Jacobsson A, Rehnmark S, and Cannon B. Norepinephrine as a morphogen?—its unique interaction with brown adipose tissue. Int J Develop Biol 39: 827–837, 1995.
  40. Ohno T, Ohinata H, Ogawa K, and Kuroshima A. Fatty acid profiles of phospholipids in brown adipose tissue from rats during cold acclimation and repetitive intermittent immobilization: with special reference to docosahexaenoic acid. Jpn J Physiol 46: 265–270, 1996.[CrossRef][Web of Science][Medline]
  41. Ortiz-Caro J, Montiel F, Pascual A, and Aranda A. Modulation of thyroid hormone nuclear receptors by short-chain fatty acids in glial C6 cells. Role of histone acetylation. J Biol Chem 261: 13997–14004, 1986.[Abstract/Free Full Text]
  42. Palosaari P and Hiltunen J. Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities. J Biol Chem 265: 2446–2449, 1990.[Abstract/Free Full Text]
  43. Raclot T. Selective mobilization of fatty acids from adipose tissue triacylglycerols. Prog Lipid Res 42: 257–288, 2003.[CrossRef][Web of Science][Medline]
  44. Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I, Shan B, Brown MS, Goldstein JL, and Mangelsdorf DJ. Regulation of mouse sterol regulatory element-binding protein-1c gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta. Genes Dev 14: 2819–2830, 2000.[Abstract/Free Full Text]
  45. Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, Schwendner S, Wang S, Thoolen M, Mangelsdorf DJ, Lustig KD, and Shan B. Role of LXRs in control of lipogenesis. Genes Dev 14: 2831–2838, 2000.[Abstract/Free Full Text]
  46. Senault C, Hlusko MT, and Portet R. Effects of diet and cold acclimation on lipid composition of rat interscapular brown adipose tissue. Ann Nutr Alim 29: 67–77, 1975.
  47. Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS, and Goldstein JL. Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest 98: 1575–1584, 1996.[Web of Science][Medline]
  48. Shimano H, Horton JD, Shimomura I, Hammer RE, Brown MS, and Goldstein JL. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells. J Clin Invest 99: 846–854, 1997.[Web of Science][Medline]
  49. Shimomura I, Hammer RE, Richardson JA, Ikemoto S, Bashmakov Y, Goldstein JL, and Brown MS. Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev 12: 3182–3194, 1998.[Abstract/Free Full Text]
  50. Stulnig TM, Oppermann U, Steffensen KR, Schuster GU, and Gustafsson JA. Liver X receptors downregulate 11beta-hydroxysteroid dehydrogenase type 1 expression and activity. Diabetes 51: 2426–2433, 2002.[Abstract/Free Full Text]
  51. Tontonoz P, Kim JB, Graves RA, and Spiegelman BM. ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation. Mol Cell Biol 13: 4753–4759, 1993.[Abstract/Free Full Text]
  52. Tvrdik P, Asadi A, Kozak LP, Nedergaard J, Cannon B, and Jacobsson A. Cig30, a mouse member of a novel membrane protein gene family, is involved in the recruitment of brown adipose tissue. J Biol Chem 272: 31738–31746, 1997.[Abstract/Free Full Text]
  53. Tvrdik P, Westerberg R, Silve S, Asadi A, Jakobsson A, Cannon B, Loison G, and Jacobsson A. Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids. J Cell Biol 149: 707–717, 2000.[Abstract/Free Full Text]
  54. Wakil SJ, Stoops JK, and Joshi VC. Fatty acid synthesis and its regulation. Ann Rev Biochem 52: 537–579, 1983.[CrossRef][Web of Science][Medline]
  55. Wang X, Sato R, Brown MS, Hua X, and Goldstein JL. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell 77: 53–62, 1994.[CrossRef][Web of Science][Medline]
  56. Watkins SM, Lin TY, Davis RM, Ching JR, DePeters EJ, Halpern GM, Walzem RL, and German JB. Unique phospholipid metabolism in mouse heart in response to dietary docosahexaenoic or alpha-linolenic acids. Lipids 36: 247–254, 2001.[Web of Science][Medline]
  57. Westerberg R, Tvrdik P, Undén AB, Månsson JE, Norlén L, Jakobsson A, Holleran WM, Elias PM, Asadi A, Flodby P, Toftgård R, Capecchi MR, and Jacobsson A. Role for ELOVL3 and fatty acid chain length in development of hair and skin function. J Biol Chem 279: 5621–5629, 2004.[Abstract/Free Full Text]
  58. Wilcke M and Alexson S. Differential induction of peroxisomal populations in subcellular fractions of rat liver. Biochim Biophys Acta 1544: 358–369, 2001.[CrossRef][Medline]
  59. Yang T, Espenshade PJ, Wright ME, Yabe D, Gong Y, Aebersold R, Goldstein JL, and Brown MS. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110: 489–500, 2002.[CrossRef][Web of Science][Medline]
  60. Yokoyama C, Wang X, Briggs MR, Admon A, Wu J, Hua X, Goldstein JL, and Brown MS. SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell 75: 187–197, 1993.[CrossRef][Web of Science][Medline]
  61. Zhang K, Kniazeva M, Han M, Li W, Yu Z, Yang Z, Li Y, Metzker ML, Allikmets R, Zack DJ, Kakuk LE, Lagali PS, Wong PW, MacDonald IM, Sieving PA, Figueroa DJ, Austin CP, Gould RJ, Ayyagari R, and Petrukhin K. A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nat Genet 27: 89–93, 2001.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
J. A. Jorgensen, D. Zadravec, and A. Jacobsson
Norepinephrine and rosiglitazone synergistically induce Elovl3 expression in brown adipocytes
Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1159 - E1168.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/4/E517    most recent
00045.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jakobsson, A.
Right arrow Articles by Jacobsson, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jakobsson, A.
Right arrow Articles by Jacobsson, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.