The gene encoding acyl-CoA-binding protein is subject to metabolic regulation by both sterol regulatory element-binding protein and peroxisome proliferator-activated receptor alpha in hepatocytes.

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular lipid-binding protein that transports acylCoA esters. The protein is expressed in most cell types at low levels; however, expression is particularly high in cells with a high turnover of fatty acids. Here we confirm a previous observation that ACBP expression in rodent liver is down-regulated by fasting, and we show that insulin but not glucose is the inducer of ACBP expression in primary rat hepatocytes. In keeping with the regulation by insulin, we show that ACBP is a sterol regulatory element-binding protein 1c (SREBP-1c) target gene in hepatocytes. Members of the SREBP family activate the rat ACBP gene through binding sites for SREBP and the auxiliary factors Sp1 and nuclear factor Y in the proximal promoter. In addition, we show that ACBP is a peroxisome proliferator-activated receptor (PPAR) alpha target gene in cultured hepatocytes and is induced in the liver by fibrates in a PPARalpha-dependent manner. Thus, ACBP is a dual PPARalpha and SREBP-1c target gene in hepatocytes. Fasting leads to reduced activity of SREBP but increased activity of PPARalpha in hepatocytes, and in keeping with ACBP being a dual target gene, we show that ACBP expression is significantly lower in livers from PPARalpha knock-out mice than in livers from wild type mice. In conclusion, expression of ACBP in rodent hepatocytes is subject to dual metabolic regulation by PPARalpha and SREBP-1c, which may reflect the need for ACBP during lipogenic as well as lipo-oxidative conditions.

The acyl-CoA-binding protein (ACBP) 1 is an intracellular lipid-binding protein that selectively binds medium and long chain acyl-CoA esters (C 14 -C 22 ) (1, 2) with high specificity and affinity (K D 1-10 nM) (3). The protein has been found in all eukaryotes investigated to date and is structurally and functionally highly conserved through evolution (4). Based on the high conservation of the protein and a large number of in vitro studies, ACBP is believed to play an important role in intracellular acyl-CoA transport and pool formation (5). However, the exact role of ACBP in lipid metabolism is still unknown. Disruption of the ACBP gene in yeast indicated that ACBP is involved in the synthesis of very long chain fatty acids and is required for proper protein sorting and vesicular trafficking (6). The mammalian ACBP gene is a typical housekeeping gene (7), although the level of ACBP differs markedly among different cell types. Cell types with a high level of ACBP expression include hepatocytes, steroidogenic cells, and adipocytes (8 -10).
Several observations suggest that the mammalian ACBP gene is subject to metabolic regulation. Thus, it was reported that fasting of rats resulted in a modest but significant decrease in the level of ACBP expression in the liver, whereas a high fat diet induced ACBP expression (11,12). These results suggest that increased ACBP expression may be linked to lipid accumulation. In keeping with that, ACBP expression is drastically induced during adipocyte differentiation of 3T3-L1 preadipocytes (13). This induction is most likely mediated, at least in part, by the peroxisome proliferator-activated receptor ␥ (PPAR␥). We have recently identified a functional peroxisome proliferator response element (PPRE) in intron 1 of the human, rat, and mouse ACBP genes and have shown by chromatin immunoprecipitation that this element binds PPAR␥ in adipocytes (10).
ACBP expression has also been reported to be induced in the liver of rodents in response to administration of peroxisome proliferators, suggesting that ACBP is a PPAR␣ target gene. In keeping with that, we have shown that PPAR␣ in transient transfections is able to activate ACBP expression through the intronic PPRE (10). It remains to be shown, however, whether PPAR␣ is indeed involved in the induction of ACBP expression by peroxisome proliferators in vivo. As ACBP expression is reported to be repressed by fasting, ACBP does not appear to belong to the group of typical PPAR␣ target genes, which are known to be induced by fasting.
The molecular mechanisms underlying the regulation of ACBP expression in hepatocytes are presently unknown. If ACBP expression is indeed repressed by fasting, it appears likely that it, by analogy with other genes, would be under the control of transcription factors activated by either glucose and/or insulin. A sterol regulatory element (SRE) has been identified in the proximal promoter of the human ACBP gene. This SRE mediates activation by the sterol regulatory elementbinding protein 1a (SREBP-1a) and confers sterol repression and androgen activation to the human proximal acbp promoter in transient transfections of the human prostate adenocarcinoma cell line LNCaP (14). SREBP-1a is only expressed at low levels in hepatocytes in vivo (15); however, another member of the SREBP family, SREBP-1c, has been shown to be necessary for induction of lipogenic genes in rat hepatocytes in response to insulin (16,17). Therefore, we hypothesized that SREBP-1c might play a role in the regulation of the rodent ACBP gene in hepatocytes in response to fasting and feeding.
In keeping with this hypothesis, we show here that insulin and not glucose induces ACBP expression in primary rat hepatocytes. Furthermore, we show that the proximal SRE is indeed functionally conserved in the rat ACBP gene and can mediate transactivation by all members of the SREBP family, including SREBP-1c. Transient transfection of hepatocytes with promoter-reporter constructs encompassing Ϫ2310 bp upstream of the rat ACBP translation start site and the entire exon 1 and intron 1 shows that activation by SREBPs is strictly dependent on this SRE. These results strongly indicate that the SRE in the proximal promoter is indeed mediating insulin activation of the ACBP gene in vivo. Detailed dissection of the sequence surrounding the SRE identified upstream binding sites for NF-Y and Sp1, respectively, both of which are necessary for optimal induction by SREBPs. Finally, we show that ACBP is a PPAR␣ target gene in hepatocytes in vivo and that disruption of the PPAR␣ gene leads to further reduction of ACBP expression in the liver during fasting. These results indicate that the ACBP gene during fasting is subject to opposing metabolic regulation by SREBP and PPAR␣, respectively.

Materials-Minimum
Eagle's medium, Schneider's medium, Dulbecco's modified Eagle's medium/F-12 (1:1), fetal calf serum, sodium pyruvate, antibiotics, and nonessential amino acids were purchased from Invitrogen. Insulin, glucose, and insulin-transferrin-sodium selenite media supplement were purchased from Sigma. Gene-specific primers for real time PCR were purchased from DNA Technology A/S (Denmark). BigDye TM Terminator cycle sequencing kit and 2ϫ SYBR Green PCR Master Mix were from Applied Biosystems (Denmark). Lysis buffer for harvesting cells after transfection was from Tropix (Denmark). Fenofibrate and ciprofibrate the were kind gifts of Drs. A. Edgar (Laboratoires Fournier, Daix, France) and M. Riteco (Sanofi, Winthrop, Maassluis, The Netherlands), respectively.
Animal Experiments-For the WT fasting/refeeding experiment, 20 C57BL/6JBom male mice, 9 weeks old, were divided at random into four groups with five individuals/group. The mice were kept at a 12-h light/ dark cycle (6.30 a.m. to 6.30 p.m.) at 22°C with free access to food and water. Following 2 weeks on a standard chow diet (Altromin 1324), the experiment was initiated in the morning at the beginning of the 12-h light cycle. Food was withdrawn from three of the groups. One group continued on the Altromin 1324 diet during the experiment (control group). After 24 h of fasting, one fasting group was sacrificed. The remaining two fasted groups were given a high carbohydrate (64.5%)/ very low fat (0.083%) diet (Altromin C1056). One of these groups was sacrificed after 12 h of refeeding on the high carbohydrate/very low fat diet, and the other group was sacrificed after 24 h of refeeding on the high carbohydrate/very low fat diet. The control group was sacrificed in the morning at the end of the experiment. Liver tissue samples were collected from all animals, frozen in liquid nitrogen, and stored at Ϫ80°C. The fasting experiment using PPAR␣ KO and WT mice was performed similarly. One group of mice of each genotype (WT, n ϭ 5; KO, n ϭ 4) was subjected to 24 h of fasting initiated in the morning. After 24 h of fasting, the mice were killed, and liver tissue samples were frozen in liquid nitrogen and stored at Ϫ80°C until RNA purification could be performed. Fed mice of each genotype were sacrificed in the morning (WT, n ϭ 4; KO, n ϭ 3).
For fibrate treatment, male 129/SvPas homozygous PPAR␣ KO and WT mice (10 -12 weeks of age) were fed for 17 days with either a standard mouse chow or one containing 0.2% (w/w) fenofibrate or 0.05% (w/w) ciprofibrate. At the end of the treatment period, each group of animals (n ϭ 7) was fasted for 4 h and killed. Livers were frozen in liquid nitrogen and stored at Ϫ80°C until RNA preparation.
Primary rat hepatocytes were isolated by collagenase perfusion from livers of Wistar rats (18). Hepatocytes (cell viability higher than 85% by trypan blue exclusion test) were cultured as a monolayer in serum-free William's E medium supplemented with 2 mM glutamine, 25 g/ml gentamycin, 100 nM dexamethasone, and 0.1% fatty acid-free bovine serum albumin at 37°C in a humidified atmosphere of 5% CO 2 , 95% air. After 4 h of incubation, medium was changed to glucose-free Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mM glutamine, 25 g/ml gentamicin, 100 nM dexamethasone, and 5.5 mM Dglucose. After overnight culture, cells were incubated for the indicated time in fresh culture medium supplemented with 2 mM glutamine, 25 g/ml gentamicin, 100 nM dexamethasone, and the indicated concentrations of D-glucose and insulin.
RNA Purification and Real Time qPCR-Total RNA was purified, and cDNA was synthesized and quantified by real time qPCR using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Each PCR contained, in a final volume of 25 l, 1 l of first-strand cDNA, 12.5 l of 2ϫ SYBR Green PCR Master Mix, and 5 pmol of each primer. All reactions were performed using the following cycling conditions: 50°C for 2 min, 95°C for 10 min followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. PCR was carried out in 96-well plates and in duplicate. Primers for real time PCR were designed using Primer Express 2.0 (Applied Biosystems). Target gene mRNA expression was normalized to TFIIB or TBP mRNA expression, and the relative amounts of all mRNAs were calculated.
Western Blotting-Whole cell extracts from cell cultures were prepared as described previously (20). Tissue was homogenized in a lysis buffer with similar composition. All protein extracts were treated with Benzonase® (Merck). Protein concentrations were determined using Bio-Rad Protein Assay (Bradford reagent). Equal amounts of protein (100 g) from animals/cells treated similarly were pooled. From these stocks, 75 g of protein was separated on 15% SDS-polyacrylamide mini gels. Protein was blotted onto polyvinylidene difluoride membranes (Micron Separation) and stained with Amido Black to confirm equal loading. Membranes were blocked in phosphate-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20 (Sigma). Primary antibodies used were rabbit anti-rat ACBP (1:1500) and rabbit anti-TFIIB (C-18, Santa Cruz Biotechnology) (1:1000). The secondary antibody used was horseradish peroxidase-conjugated swine anti-rabbit IgG (1:1000) (Dako). Enhanced chemiluminescence (Amersham Biosciences) was used for detection.
Transient Transfections-HepG2 and SL2 cells were transfected at 50 -70% confluence in 12-well plates using the DC-Chol lipofection procedure (26) and a total of 1 g of DNA/well. Following 5-6 h of incubation with DNA mixture, the medium was replaced, and cells were harvested 20 h later in lysis buffer (Tropix), and the lysates were stored at Ϫ80°C. All transfections were performed as triplicates. Luciferase and ␤-galactosidase assays were performed as described previously (27).
Adenoviral Vectors and Transduction of AML-12 Cells-Recombinant adenoviruses were constructed by subcloning the respective cDNA (SREBP-1c) or full-length (mouse PPAR␣) sequence into pShuttle and recombining these constructs with adenoviral vectors using the AdEasy cloning system from Stratagene (AH-diagnostic, Denmark). The constitutive active rat SREBP-1c cDNA was derived from pSVSPORT-ADD1-403 (28) and used to make Ad-CA-SREBP-1c. Viruses were amplified in HEK293 cells and purified by using CsCl ultracentrifugation followed by desalting on PD10 columns (Amersham Biosciences). Titers of adenoviruses were initially determined by plaque assay, and equal viral concentrations in the different experiments were subsequently verified by determining expression of the adenoviral transcript AdE4 normalized to TFIIB expression by real time qPCR. Cells were transduced by adding the viruses directly to the media. Media containing the viruses were removed after 2 h, and new media were added for further incubation (22 h).
Statistical Analysis-Data from the feeding experiments were analyzed by one-way analysis of variance using the Statistical Analysis System (SAS) Analyst application (SAS release 8.02 (1999 -2001) by SAS Institute Inc., Cary, NC). Results are presented as mean Ϯ S.D.

ACBP Expression Is Regulated by Fasting/Refeeding in Ro-
dents-Previous semi-quantitative studies have shown that rats that have been fasted have a decreased expression level of ACBP mRNA and protein in the liver (12) and that feeding a high fat diet increases hepatic ACBP mRNA (11), indicating that ACBP expression is metabolically regulated. To establish further how fasting and feeding regulated ACBP expression, we performed a fasting/refeeding experiment with mice. Twenty C57BL/6JBom male mice were divided randomly into four groups with five individuals per group. The control group received standard diet ad libitum throughout the study. The remaining three groups were fasted for 24 h. Subsequently the second group was sacrificed, and liver tissue was collected for RNA isolation. The remaining two fasting groups were given a high carbohydrate/very low fat diet and sacrificed after 12 and 24 h of refeeding, respectively.
As reported previously (16), hepatic mRNA levels of ACC and fatty-acid synthetase (FAS) were down-regulated by fasting and superinduced by refeeding (Fig. 1A). Regulation of FAS activity during fasting/refeeding is known to take place primarily at the transcriptional level (reviewed in Ref. 29), whereas ACC activity is regulated transcriptionally as well as posttranslationally (reviewed in Ref. 30). The transcriptional effects are supposed to be mediated by a combined effect of insulin-induced activation of SREBP-1c and glucose-induced activation of the carbohydrate-response element-binding protein (ChREBP) (31,32). SREBP-1c itself is known to be induced by refeeding, both at the mRNA and at the protein levels (16). In keeping with this, the regulation of SREBP-1c mirrored the regulation of FAS and ACC (Fig. 1A). Expression of ACO is known to be induced in response to PPAR␣ activation (16), and this regulation is reflected in similar changes in protein expression (33). In keeping with this, ACO expression was significantly induced by fasting and was down-regulated by refeeding. Stearoyl-CoA desaturase 1 (SCD-1), which is an SREBP-1c (34) and a PPAR␣ (35) target gene, but which has not been reported to be regulated by ChREBP, was more modestly down-regulated by fasting. The induction of SCD-1 by refeeding was less dramatic and not observed until after 24 h of refeeding. Hepatic ACBP mRNA level was modestly but significantly reduced in mice fasted for 24 h compared with ad libitum fed control animals (Fig. 1A), and similar to SCD-1 expression, 24 but not 12 h of refeeding reestablished ACBP expression. A similar regulation of ACBP protein expression was observed (Fig. 1B).

ACBP Expression Is Induced by Insulin but Not by Glucose in
Primary Rat Hepatocytes-To investigate whether the regulation of ACBP expression in hepatocytes by fasting/refeeding is mediated by glucose and/or insulin, we treated freshly isolated primary rat hepatocytes with either increasing concentrations of insulin ( Fig. 2A) or increasing concentrations of glucose (Fig.  2B). These experiments clearly demonstrated that both ACBP and SREBP-1c expressions are induced by insulin ( Fig. 2A) and not by glucose (Fig. 2B) in rat hepatocytes. The activation of SREBP-1c expression by insulin but not glucose is in keeping with previous findings that insulin and not glucose is the prime inducer of SREBP-1c expression in rat hepatocytes (36,37). By contrast, expression of FAS, which is both an SREBP-1c and a ChREBP target gene (38), is activated by glucose (Fig. 2B) as well as by insulin ( Fig. 2A). Most interestingly, SCD-1 expression is not only activated by insulin ( Fig. 2A), as reported previously (39), but also by glucose (Fig. 2B), suggesting that this gene may be subject to activation by a glucose-regulated transcription factor.
These results clearly show that in rat hepatocytes ACBP groups together with SREBP-1 as an insulin-regulated gene and suggest that the transcription factor mediating this response could be SREBP-1c. In keeping with the data from the fasting/refeeding experiment, ACBP protein levels were also induced by insulin (Fig. 2C).
ACBP Expression Is Activated by SREBP-1c in Hepatocytes-To investigate whether ACBP is an SREBP-1c target gene in hepatocytes, we transduced the murine AML-12 hepatocyte cell line with an adenoviral vector expressing the constitutive active SREBP-1c (Ad-CA-SREBP-1c) or an empty adenoviral vector (Ad-Vector). As shown in Fig. 3, SREBP-1c significantly and dose-dependently increased ACBP expression both at the mRNA as well as the protein level.
The Proximal Promoter of the Rat ACBP Gene Mediates Activation by All SREBP Subtypes-The proximal promoter of the rat ACBP gene contains a potential SRE and NF-Y-RE, which on a sequence basis are well conserved between rodents and humans (Fig. 4).
To determine whether the proximal promoter can mediate transactivation by members of the SREBP family, we transiently transfected HepG2 cells with a proximal promoter-reporter construct (pluc-rACBP(Ϫ182/ϩ1)) together with increasing concentrations of a plasmid encoding either the constitutive active SREBP-1a, SREBP-1c, or SREBP-2 protein. As shown in Fig. 5, the rat ACBP(Ϫ182/ϩ1) is activated in a dose-dependent manner by all SREBP subtypes in HepG2 cells. In keeping with the longer and more powerful transactivation domains in SREBP-1a and SREBP-2 compared with SREBP-1c (40), the 1a and 2 subtypes appear to be more efficient activators of the acbp promoter in transient transfections than the 1c subtype.

Dissection of the Proximal Promoter of the Rat ACBP-In
addition to the potential SRE at position Ϫ129, we identified another potential SRE at position Ϫ172. Most interestingly, the latter overlaps with a potential Sp1-binding site (Fig. 4). Both NF-Y and Sp1 have been reported as auxiliary transcription factors necessary for optimal transactivation by the SREBPs (41,42).
To investigate which of the potential SREs were responsible for mediating the activation by SREBPs and to further investigate which other auxiliary sites were required, we generated a series of reporter constructs containing various mutations and deletions of the rat acbp promoter linked to the luciferase reporter gene. As shown in Fig. 6, mutation of the Ϫ129 SRE significantly decreased, but did not totally block, activation by SREBPs. In contrast, mutation of the Ϫ129 SRE as well as deletion of the Ϫ172 SRE totally blocked activation by SREBPs, suggesting that the Ϫ172 SRE is able to function as a weak SRE in transient transfections. Mutation of the putative NF-Y-response element also completely abolished the transactivation by the SREBPs, whereas deletion of the potential SRE/Sp1-binding site at Ϫ172 decreased promoter activity but did not abolish the induction by SREBPs. These results demonstrate that the putative NF-Y site is necessary for SREBP transactivation in HepG2 cells and that the SRE site located at Ϫ129 is the main responsible SRE for conferring SREBP responsiveness to the rat acbp proximal promoter. In addition, the results suggest that the upstream SRE/Sp1-binding site can mediate weak transactivation by SREBPs.
To evaluate whether the potential SRE/Sp1 site could be a functional Sp1-binding site, co-transfection studies were performed in Drosophila SL2 cells. This cell line is devoid of endogenous Sp1 and therefore makes an ideal assay system for investigating Sp1 transactivation (25). The promoter-reporter constructs with or without the SRE/Sp1 site were transfected into SL2 cells along with either pPac or pPac-Sp1. Co-transfection of pPac-Sp1 resulted in a severalfold induction of the luciferase activity from the pluc-rACBP(Ϫ182/ϩ1) reporter construct, whereas promoter activity was nearly abolished when the SRE/Sp1 site was deleted (Fig. 7A). This strongly indicates that Sp1 can bind to and transactivate the acbp promoter through the SRE/Sp1-binding site.
To investigate the synergy between SREBP, NF-Y, and Sp1 transcription factors on the acbp promoter, we co-transfected SL2 cells with the pluc-rACBP(Ϫ182/ϩ1) reporter construct and combinations of expression vectors for the individual transcription factors (Fig. 7B). All SREBPs synergized with Sp1 as well as with NF-Y; however, the combination of an SREBP subtype with both Sp1 and NF-Y gave the highest transactivation.
The Proximal SRE Is Functional in a Larger Promoter Fragment-Next we wanted to investigate whether SREBP transactivation of the rat acbp promoter was conserved in the context of a larger fragment of the ACBP locus, and whether the proximal SRE was necessary for this transactivation. A reporter-promoter construct containing both upstream sequences and the entire exon 1 and intron 1 (pluc-rACBP(Ϫ2310/ϩ979)) was used for transient transfections together with SREBP-1a, SREBP-1c, or SREBP-2 expression vectors. As shown in Fig. 8, all members of the SREBP family activate transcription from the rat acbp promoter in this construct. Furthermore, mutation of the Ϫ129 SRE site almost completely abolishes the transactivation by members of the SREBPs, demonstrating that this SRE site is necessary for SREBP transactivation of the rat ACBP gene.

Induction of ACBP Expression by Peroxisome Proliferators Is Dependent on PPAR␣-
The finding that the rodent ACBP gene is an SREBP target gene and is down-regulated by fasting and induced by insulin appears at odds with results suggesting that the rodent ACBP gene is a PPAR␣ target gene. We have recently identified a PPRE in intron 1 of the rat, mouse, and human ACBP gene, and we have shown that this element mediates PPAR␥ activation of the acbp promoter in adipocytes. In addition, we showed that at least in transient transfections, PPAR␣ is able to activate the rat and human acbp promoters through this PPRE in fibroblasts (10). These results are in keeping with previous results showing that perfluorodecanoic acid (11) and the 3-thia fatty acid tetradecyl-thioacetic acid (43) induce ACBP mRNA and protein in rat liver. However, because these compounds are not specific activators of PPAR␣, it could not be conclusively determined whether ACBP was a PPAR␣ target gene in vivo.
In keeping with the notion that ACBP is a PPAR␣ target gene, murine AML-12 hepatocytes transduced with an adenoviral vector expressing PPAR␣ (Ad-PPAR␣) had significantly higher expression of ACBP than control cells (Fig. 9A). To investigate the importance of PPAR␣ in fibrate regulation of ACBP expression in rodent liver, PPAR␣ϩ/ϩ and PPAR␣Ϫ/Ϫ 129/SvPas mice were given a daily dose of ciprofibrate or fenofibrate for 17 days, and RNA was purified from liver. The results presented in Fig. 9B show that ACBP is induced 3-fold by these fibrates and that this induction is strictly dependent on PPAR␣. In contrast, PPAR␣ does not seem to influence the basal level of ACBP expression. Thus, despite the decrease in ACBP expression during fasting, ACBP is a PPAR␣ target gene.
Opposing Regulation of ACBP Expression by SREBP and PPAR␣ in Hepatocytes-The finding that the ACBP gene is both a PPAR␣ and an SREBP-1c target gene suggests that transcription from the ACBP locus would be subject to dual and opposing regulatory forces under fasting conditions. Thus, during fasting PPAR␣ would be expected to activate ACBP expression, whereas the activation by SREBP-1c would be dramatically reduced due to the decreased level of insulin and glucose. If this assumption were correct, lack of PPAR␣ would be expected to lead to a more pronounced decrease in ACBP expression during fasting.
To test this notion, we fasted PPAR␣ϩ/ϩ and PPARϪ/Ϫ C57BL/6J mice for 24 h, and we compared the effect of fasting on liver mRNA expression in the two groups of mice. As reported previously (44), disruption of PPAR␣ expression interfered with normal regulation of FAS and ACC in the fed liver. The expression of SREBP-1 target genes including FAS, ACC, glucokinase, and SREBP-1c itself was significantly decreased in the PPAR␣ KO compared with the WT mice ( Fig. 10A and results not shown). However, in keeping with what was found in the experiment reported in Fig. 9, disruption of PPAR␣ did not affect ACBP expression in the fed liver (Fig. 10A). Similarly, the expression of other SREBP-1c target genes like lipoprotein lipase and ⌬6 desaturase were not affected by the disruption of the PPAR␣ gene (data not shown). In addition, as reported previously (45), disruption of PPAR␣ did not affect expression of the PPAR␣ target gene ACO in the fed state (Fig.  10A). Upon fasting, levels of ACC, FAS, glucokinase, and SREBP-1c mRNA were reduced to similar levels in livers of PPAR␣ϩ/ϩ and PPAR␣Ϫ/Ϫ mice (Fig. 10A and results not shown). In contrast, expression of ACO was increased in WT mice but decreased in PPAR␣ KO mice, clearly showing that the induction by fasting was dependent on PPAR␣. In keeping with our data showing that ACBP is both an SREBP-1c and a PPAR␣ target gene, ACBP expression was down-regulated by fasting and was significantly lower in livers of fasted PPAR␣ KO animals compared with fasted WT animals (Fig. 10A). These changes in ACBP mRNA expression were reflected in similar changes in protein expression (Fig. 10). DISCUSSION The mammalian ACBP gene belongs to the group of typical housekeeping genes but is nevertheless differentially expressed in different cell types. Cell types with a considerable lipid metabolism, e.g. adipocytes and hepatocytes, have high levels of ACBP expression. We have shown previously that ACBP is significantly induced during adipocyte differentiation (13) and is a direct PPAR␥ target gene in adipocytes (10). In this report we have investigated the transcriptional regulation of ACBP expression in hepatocytes. Previous results showing that ACBP is induced in hepatocytes in response to peroxisome proliferators (11,43) and that the acbp promoter is activated by PPAR␣ in transient transfections (10) have suggested that ACBP is a PPAR␣ target gene. However, other reports claiming a down-regulation of ACBP expression by fasting (11,12) suggest that ACBP does not behave as a typical PPAR␣ target gene. We have reinvestigated the effect of fasting and have shown that ACBP mRNA expression is indeed modestly down-regulated by fasting for 24 h and up-regulated again following refeeding with a carbohydrate-rich diet for 24 h. In contrast, the PPAR␣ target gene ACO is significantly induced by fasting. As reported by others, the SREBP-1c and ChREBP target genes ACC and FAS as well as SREBP-1c itself were repressed by fasting and super-induced by refeeding a carbohydrate-rich diet (Fig. 1). By using rat primary hepatocytes, we show that insulin but not glucose induces ACBP mRNA expression, indicating that the prime inducer of ACBP during feeding is insulin. In keeping with previous reports (36 -38), expression of SREBP-1c is also induced by insulin and not by glucose in rat hepatocytes, whereas FAS was induced by both glucose and insulin (Fig. 2).
SREBP-1c is known to be essential for activation of lipogenic genes by insulin in hepatocytes, and we therefore investigated whether the rat ACBP gene is an SREBP-1c target gene. An SRE has previously been reported in the human ACBP gene, where it was shown to mediate transactivation by SREBP-1a and to confer androgen activation of the human acbp proximal promoter in a prostate adenocarcinoma cell line. Androgens are known to induce lipogenesis in these cells by SREBP-dependent mechanisms (46). By using adenoviral vectors to ectopically express a constitutive active SREBP-1c, we showed that ACBP is indeed an SREBP-1c target gene in hepatocytes (Fig. 3).
We therefore proceeded to identify and characterize the DNA elements responsible for mediating the transactivation by SREBP-1c and other members of the SREBP family in hepatocytes. We showed that the proximal promoter of the rat ACBP gene contains a functional SRE and auxiliary NF-Y and Sp1 sites, and that all members of the SREBP family activate the promoter in synergy with NF-Y and Sp1 (Fig. 7). In HepG2 cells NF-Y is the most important auxiliary factor, and Sp1 appears to play a very limited role (Fig. 6). In a similar dissection of the SRE in the proximal promoter of FAS, Osborne and co-workers (41) reported that all subtypes of SREBP were dependent on NF-Y, whereas only SREBP-1c showed a strong dependence of Sp1. We do not see such a difference between the SREBP subtypes either in SL2 cells or in HepG2 cells (Figs. 6 and 7), indicating that the requirements for auxiliary factors are promoter-dependent rather than subtype-dependent. In addition, the relative importance of the factors appears to be cell typespecific as mutation of the Sp1 site reduces activation of the promoter by endogenous transcription factors to background levels in the pancreatic ␤-cell line INS-1 (data not shown). Most importantly, when investigated in the context of 2310 bp of upstream flanking sequence and the entire exon 1 and intron 1 (a total of 3289 bp), the proximal SRE was necessary for SREBP activation of the acbp promoter (Fig. 8). Thus, the proximal SRE is the only functional SRE in a construct encompassing a very large fraction of the ACBP locus. Most interestingly, the functionality of the SRE in these large constructs required the presence of the intron 1 (results not shown), suggesting that SREBPs synergize with transcription factors binding to the intronic region. As we have characterized previously for a well conserved PPRE in intron 1, we speculated that SREBP might synergize with factors binding to this direct repeat-1. However, mutation of the PPRE in intron 1 did not interfere with the synergy (results not shown).
The finding that ACBP is down-regulated by fasting necessitated in vivo confirmation that ACBP is indeed a PPAR␣ target gene. We therefore transduced AML-12 cells with an adenoviral vector expressing PPAR␣, and we showed that ACBP expression was significantly induced by PPAR␣ in this hepatocyte cell line. To verify that ACBP is indeed a PPAR␣ target gene in vivo, we treated WT and PPAR␣ KO mice with PPAR␣-specific ligands. Our results showed that induction of ACBP expression by these ligands was strictly dependent on the expression of PPAR␣. Thus, ACBP is a dual PPAR␣ and SREBP target gene in hepatocytes. This finding has implications for the regulation of ACBP expression during fasting where the acbp promoter would be expected to be subject to dual and opposing forces by the decreased SREBP-1c activity and the increased PPAR␣ activity. To investigate if such an antagonism exists, we subjected WT and PPAR␣ KO mice to a 24-h fasting period, and we determined the effect on gene expression in the liver. Our results showed that ACBP expression is significantly lower in livers of fasted PPAR␣ KO mice than in livers of WT mice, thereby corroborating the notion that PPAR␣ counteracts the repression by the decreased SREBP-1c activity. By contrast, the expression of all other SREBP target genes investigated did not differ significantly between WT and PPAR␣ animals in the fasted state. Most interestingly, however, a large number of SREBP-1c target genes were expressed at significantly lower levels in PPAR␣ KO compared with WT animals in the fed state. This finding extends previous results showing that FAS and ACC were deregulated and expressed at lower levels in livers of PPAR␣ KO compared with WT animals (44). As LXR␣ is a PPAR␣ target gene (47,48), and LXR␣ activates the srebp-1c promoter (49), we speculated that decreased expression of LXR␣ in the PPAR␣ KO animals in the fed state could explain the finding. However, in the fed state LXR␣ mRNA levels were not affected by the disruption of the PPAR␣ gene (results not shown).
ACBP is not the first dual PPAR␣ and SREBP-1c target gene to be reported. Other members of this family include lipoprotein lipase (50,51), SCD-1 (34,35), and the ⌬5and ⌬6-desaturases (52). However, expression of lipoprotein lipase and the desaturases was neither significantly affected by fasting nor by disruption of PPAR␣ (results not shown), suggesting that transcription factors other than SREBP-1c and PPAR␣ are ratelimiting under these conditions. SCD-1 expression follows that FIG. 11. Model for the dual regulation of ACBP expression by PPARs and SREBPs. In rodent hepatocytes the ACBP gene is regulated by insulin via the induction of SREBP-1c activity. SREBP-1c activates expression by binding to an SRE in the proximal promoter, and the transcription factors NF-Y and Sp1 function as auxiliary factors. PPAR␣/RXR activates ACBP expression in response to fibrates and other PPAR␣ activators by binding to the PPRE in intron 1. Fasting leads to inhibition of SREBP-1c activity but induces PPAR␣ activity. In adipocytes and other cells that express PPAR␥, ACBP expression is activated by thiazolidinediones (TZDs) and other PPAR␥ activators through PPAR␥/RXR binding to the intronic PPRE. Position of regulatory elements and 5Ј end of exons relative to the translation start site are indicated in italics. See text for further details. of other SREBP-1c target genes, i.e. it is only affected by disruption of the PPAR␣ gene in the fed state, indicating that SREBP-1c is much more important than PPAR␣ in the regulation of SCD-1 expression in the liver during fasting/refeeding. Similarly, regulation of ACBP expression during fasting/ refeeding is dominated by SREBP-1c; however, by contrast to the other dual PPAR␣ and SREBP-1c target genes we have looked at, ACBP shows a significant dependence on PPAR␣ in the fasted state.
Our results on the regulation of the rodent ACBP gene by SREBP-1c and PPAR␣ and -␥ are summarized in Fig. 11. In adipocytes ACBP expression is induced by the lipogenic transcription factor PPAR␥. Whether ACBP is also an SREBP-1 target gene in adipocytes remains to be shown, but it is clear that ACBP is associated with lipogenesis. In hepatocytes, regulation appears to be dominated by the lipogenic transcription factor SREBP-1c. However, ACBP expression is also induced by the lipo-oxidative transcription factor PPAR␣, which in the case of fasting counteracts the dominant regulation by SREBP-1c. In other situations where PPAR␣ is activated, e.g. by fibrates, ACBP expression is induced. This dual regulation of ACBP expression by a lipogenic as well as a lipo-oxidative transcription factor in hepatocytes may reflect the fact that ACBP is an intracellular acyl-CoA carrier. It appears likely that there would be an increased demand for an acyl-CoA donor/carrier under both lipogenic and lipo-oxidative conditions. However, a full understanding of this dual regulation awaits insight into the biochemical function of ACBP in vivo.