Torben Helledie

The Orphan Nuclear Receptor Rev-Erbα Is a Peroxisome Proliferator-activated Receptor (PPAR) γ Target Gene and Promotes PPARγ-induced Adipocyte Differentiation

Rev-Erbα (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor α gene. Rev-Erbα mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbα expression is induced by PPARγ activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro. Furthermore, activated PPARγ induces Rev-Erbα promoter activity by binding to the direct repeat (DR)-2 response element Rev-DR2. Mutations of the 5′ or 3′ half-sites of the response element totally abrogated PPARγ binding and transcriptional activation, identifying this site as a novel type of functional PPARγ response element. Finally, ectopic expression of Rev-Erbα in 3T3-L1 preadipocytes potentiated adipocyte differentiation induced by the PPARγ ligand rosiglitazone. These results identify Rev-Erbα as a target gene of PPARγ in adipose tissue and demonstrate a role for this nuclear receptor as a promoter of adipocyte differentiation.

The orphan nuclear receptor Rev-erbα is a PPARγ target gene and promotes PPARγ-induced adipocyte differentiation

Rev-Erbα (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor α gene. Rev-Erbα mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbα expression is induced by PPARγ activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro. Furthermore, activated PPARγ induces Rev-Erbα promoter activity by binding to the direct repeat (DR)-2 response element Rev-DR2. Mutations of the 5′ or 3′ half-sites of the response element totally abrogated PPARγ binding and transcriptional activation, identifying this site as a novel type of functional PPARγ response element. Finally, ectopic expression of Rev-Erbα in 3T3-L1 preadipocytes potentiated adipocyte differentiation induced by the PPARγ ligand rosiglitazone. These results identify Rev-Erbα as a target gene of PPARγ in adipose tissue and demonstrate a role for this nuclear receptor as a promoter of adipocyte differentiation.

Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains of PPAR gamma and PPAR alpha were significantly weaker. PPAR-NCoR interactions were antagonized by ligands in the two-hybrid system, but were ligand-insensitive in in vitro pull-down assays. Interaction between PPAR delta and NCoR was unaffected by coexpression of retinoid X receptor (RXR) alpha. The PPAR delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well with interaction domains I and II of NCoR. In transient transfection experiments, NCoR and the related silencing mediator for retinoid and thyroid hormone receptor (SMRT) were shown to exert a marked dose-dependent repression of ligand-induced PPAR delta-mediated transactivation; in addition, transactivation induced by the cAMP-elevating agent forskolin was efficiently reduced to basal levels by NCoR as well as SMRT coexpression. Our results suggest that the transactivation potential of liganded PPAR delta can be fine-tuned by interaction with NCoR and SMRT in a manner determined by the expression levels of corepressors and coactivators.

Disruption of the Acyl-CoA-binding Protein Gene Delays Hepatic Adaptation to Metabolic Changes at Weaning

The acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor is an intracellular protein that binds C14–C22 acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP−/−). These mice are viable and fertile and develop normally. However, around weaning, the ACBP−/− mice go through a crisis with overall weakness and a slightly decreased growth rate. Using microarray analysis, we show that the liver of ACBP−/− mice displays a significantly delayed adaptation to weaning with late induction of target genes of the sterol regulatory element-binding protein (SREBP) family. As a result, hepatic de novo cholesterogenesis is decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors, leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads to delayed induction of the lipogenic gene program in the liver.

ACBP--a PPAR and SREBP modulated housekeeping gene.

The acyl-CoA binding protein (ACBP) is a 10 kD intracellular lipid binding protein that binds and transports acyl-CoA esters. The protein is expressed in most cell types at low levels; however, expression differs markedly between different cell types with expression being particularly high in e.g. cells with a high turnover of fatty acids. We show here that the relatively high basal promoter activity of the rat ACBP gene in fibroblasts and hepatoma cells relies on sequences between −331 to −182 and on the Sp1 and NF-Y sites at −172 and −143, respectively. The basal transcription is modulated by members of the PPAR and SREBP families. In adipocytes, PPARγ is in part responsible for the induction during adipocyte differentiation, but other transcription factors appear to play a role as well. In hepatocytes, SREBP-1c is the main regulator of ACBP in response to changes in insulin levels during fasting/refeeding. PPARα counteracts this effect by stimulating ACBP expression during fasting. In addition, PPARα mediates the induction of ACBP expression in response to peroxisome proliferators. PPARα and PPARγ do not require sequences upstream of −182 for transactivation; however, SREBP-1c requires the synergistic action of sequences in intron 1 for transactivation of the ACBP promoter.

Role of adipocyte lipid-binding protein (ALBP) and acyl-CoA binding protein (ACBP) in PPAR-mediated transactivation

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.

The acyl-CoA binding protein is required for normal epidermal barrier function in mice.

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP−/− mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP−/− mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP+/+ and ACBP−/− mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP−/− mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP−/− mice display a ∼50% increased transepidermal water loss compared with ACBP+/+ mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.