Expression In Preadipocytes Research Articles

Identification of adipocyte differentiation-related regulatory element for adrenomedullin gene repression (ADRE-AR) in 3T3-L1 cells

Adrenomedullin (AM), a potent vasodilator peptide, has been suggested to act against cardiovascular complications and insulin resistance in the metabolic syndrome. We have already reported the AM gene repression in the early phase of adipocyte differentiation of NIH 3T3-L1 cells. Here we show adipocyte differentiation-related regulatory element for AM gene repression (ADRE-AR) in 36-bp region (−2135/−2100) of the AM gene. 3T3-L1 cells were induced to differentiate to adipocytes by insulin, dexamethasone and 3-isobutyl-1-methylxanthine. On the third day of differentiation, the promoter function was analyzed using the reporter plasmids, which contain the promoter region of AM gene (−4616/+108) in pGL3-basic luciferase reporter vector. The promoter activity decreased to about 20% in 3T3-L1 adipocytes when compared with 3T3-L1 preadipocytes, and a 36-bp region (−2135 to −2100) upstream from the transcription initiation site of the AM gene was necessary for higher AM gene expression in preadipocytes. This 36-bp ADRE-AR contains three copies of G/AAAA sequence (5′- GAAAT GAAAGT AAAA-3′) (−2124/−2110), which are conserved between mouse and human, and the introduction of mutations in each copy of G/AAAA sequence decreased the promoter activity in preadipocytes and adipocytes. Electrophoretic mobility shift assay showed that the full-length ADRE-AR was specifically bound by a certain nuclear protein(s). The present study has raised the possibility that ADRE-AR may play important roles in the AM gene expression in preadipocytes, and that the AM gene may be repressed through the ADRE-AR in adipocytes.

Adipose Tissue and Circulating Endothelial Cell Specific Molecule-1 in Human Obesity

Adipocytes produce the endothelial-cell specific molecule-1 (ESM-1), which inhibits leukocyte adhesion and migration through the endothelium. This study investigates ESM-1 expression and regulation in human adipose tissue. Subcutaneous abdominal adipose tissue was obtained from seventy postmenopausal women. Fourteen women subsequently underwent non-pharmacological weight reduction. In vitro experiments were performed on adipocytes isolated from human mammary adipose tissue. We determined gene expression by TaqMan RT-PCR and measured ESM-1 levels in serum and cell culture medium by ELISA. Mature adipocytes produced ESM-1. ESM-1 gene expression was higher in adipocytes than in preadipocytes. Cortisol inhibited ESM-1 gene expression in preadipocytes. Insulin and cortisol inhibited adipocyte ESM-1 production in adipocytes. This inhibitory effect of insulin was attenuated by insulin resistance, as ESM-1 gene expression in subcutaneous adipose tissue was increased in obese, hyperinsulinemic women. In contrast, ESM-1 serum levels were reduced in obese women and inversely correlated to C-reactive protein levels. Five percent weight loss did not markedly change gene expression. Circulating ESM-1 levels increased significantly, albeit modestly. ESM-1 is actively produced by adipocytes. However, since ESM-1 adipocyte gene expression and circulating plasma levels are not correlated, other sources of ESM-1 may be more important. Circulating ESM-1 levels are reduced in the overweight and obese, consistent with the notion that ESM-1 may play some role in obesity-associated vascular disease.

Functional Analysis of Hes-1 in Preadipocytes

Notch signaling blocks differentiation of 3T3-L1 preadipocytes, and this can be mimicked by constitutive expression of the Notch target gene Hes-1. Although considered initially to function only as a repressor, recent evidence indicates that Hes-1 can also activate transcription. We show here that the domains of Hes-1 needed to block adipogenesis coincide with those necessary for transcriptional repression. HRT1, another basic-helix-loop-helix protein and potential Hes-1 partner, was also induced by Notch in 3T3-L1 cells but did not block adipogenesis, suggesting that Hes-1 functions primarily as a homodimer or possibly as a heterodimer with an unknown partner. Purification of Hes-1 identified the Groucho/transducin-like enhancer of split family of corepressors as the only significant Hes-1 interacting proteins in vivo. An evaluation of global gene expression in preadipocytes identified approximately 200 Hes-1-responsive genes comprising roughly equal numbers of up-regulated and down-regulated genes. However, promoter analyses indicated that the down-regulated genes were significantly more likely to contain Hes-1 binding sites, indicating that Hes-1 is more likely to repress transcription of its direct targets. We conclude that Notch most likely blocks adipogenesis through the induction of Hes-1 homodimers, which repress transcription of key target genes.

Krüppel-like Factor-6 Promotes Preadipocyte Differentiation through Histone Deacetylase 3-dependent Repression of DLK1

Preadipocyte differentiation occurs during distinct periods of human development and is a key determinant of body mass. Transcriptional events underlying adipogenesis continue to emerge, but the link between chromatin remodeling of specific target loci and preadipocyte differentiation remains elusive. We have identified Krüppel-like factor-6 (KLF6), a recently described tumor suppressor gene, as a repressor of the proto-oncogene Delta-like 1 (Dlk1), a gene encoding a transmembrane protein that inhibits adipocyte differentiation. Forced expression of KLF6 strongly inhibits Dlk1 expression in preadipocytes and NIH 3T3 cells in vivo, whereas down-regulation of KLF6 in 3T3-L1 cells by small interfering RNA prevents adipogenesis. Repression of Dlk1 requires HDAC3 deacetylase activity, which is recruited to the endogenous Dlk1 promoter where it interacts with KLF6. Our studies identify the interaction between HDAC3 and KLF6 as a potential mechanism underlying human adipogenesis, and highlight the role of KLF6 as a multifunctional transcriptional regulator capable of mediating adipocyte differentiation through gene repression.

Prediction of preadipocyte differentiation by gene expression reveals role of insulin receptor substrates and necdin

The insulin/IGF-1 (insulin-like growth factor 1) signalling pathway promotes adipocyte differentiation via complex signalling networks. Here, using microarray analysis of brown preadipocytes that are derived from wild-type and insulin receptor substrate (Irs) knockout animals that exhibit progressively impaired differentiation, we define 374 genes/expressed-sequence tags whose expression in preadipocytes correlates with the ultimate ability of the cells to differentiate. Many of these genes, including preadipocyte factor-1 (Pref-1) and multiple members of the Wnt signalling pathway, are related to early adipogenic events. Necdin is also markedly increased in Irs knockout cells that cannot differentiate, and knockdown of necdin restores brown adipogenesis with downregulation of Pref-1 and Wnt10a expression. Insulin receptor substrate proteins regulate a necdin-E2F4 interaction that represses peroxisome-proliferator-activated receptor gamma (PPARgamma) transcription via a cyclic AMP response element binding protein (CREB)-dependent pathway. Together these define a key signalling network that is involved in brown preadipocyte determination.

Role of fatty acids in adipocyte growth and development.

Fat is typically added to diets as a source of energy. The alternative aspects considered here are the use of specific fats to alter the fatty acid profile of adipose tissue toward creation of value-added products and the potential for individual fatty acids to alter gene expression and control adipose tissue development. Emphasis is placed on the omega-3 fatty acids, such as those found in fish oil, and on CLA. The most common association of fatty acids with adipose tissue is related to their storage as triglycerides in mature adipocytes and the consequences of excess accumulation in obesity. Fatty acids and their derivatives also can have hormone-like effects and have been be shown to regulate gene expression in preadipocytes, which ultimately effects their proliferation and differentiation. Long-chain, saturated, and polyunsaturated fatty acids have been shown to regulate transcription factors, such as CCAAT/enhancer binding protein, peroxisome proliferator activated receptor, and other adipose-specific genes, very early in adipocyte development. These effects have the potential to affect fat cell number at maturity. Specifically, there is evidence that the fatty acids in fish oil, such as docosahexaenoic and eicosopentaenoic acids, and fatty acids in the CLA series, decrease preadipocyte proliferation in cell lines and reduce adiposity in rodents. There is little direct evidence of the ability of fatty acids to manipulate adipocyte development in non-rodent species. The genetic, nutritional, and pharmacological manipulation of adipose tissue in meat animals has long been of interest to animal scientists. An understanding of the ability of fatty acids to regulate factors such as adipocyte size and number, particularly in meat animals, would be of great interest. The evidence for regulatory roles of fatty acids in development from rodent and in vitro studies and their potential application to meat animals are reviewed.

Dexamethasone reverses TGF-β-mediated inhibition of primary rat preadipocyte differentiation

Dexamethasone and transforming growth factor-β (TGF-β) show contrary effects on differentiation of adipocytes. Dexamethasone stimulates adipocyte differentiation whereas TGF-β inhibits it. In the present study, we investigated whether dexamethasone could reverse the TGF-β-mediated inhibition of preadipocyte differentiation. Primary rat preadipocytes, obtained from Sprague–Dawley rats, were pretreated with dexamethasone in the presence or absence of TGF-β, prior to the induction of differentiation. Co-treatment of dexamethasone and TGF-β before inducing differentiation reversed the TGF-β-mediated inhibition of preadipocyte differentiation. In order to elucidate the mechanism by which dexamethasone reversed the effect of TGF-β on the inhibition of preadipocyte differentiation, the expression of CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) was examined. Dexamethasone increased C/EBPα and PPARγ expression in the absence of TGF-β and also recovered the TGF-β-mediated suppression of C/EBPα expression in preadipocytes. Its effect was sustained in differentiated adipocytes as well. However, those effects were not observed in 3T3-L1 preadipocytes or differentiated adipocytes. These results indicate that dexamethasone reverses the TGF-β-mediated suppression of adipocyte differentiation by regulating the expression of C/EBPα and PPARγ, which is dependent on the cellular context.

Norepinephrine Specifically Stimulates Ribonucleotide Reductase Subunit R2 Gene Expression in Proliferating Brown Adipocytes: Mediation via a cAMP/PKA Pathway Involving Src and Erk1/2 Kinases

We have examined whether a qualitative switch occurs in the response of the ribonucleotide reductase (RNR) genes to the effect of the physiological cAMP-elevating agent norepinephrine (NE) during the development of brown adipocytes. Basal expression of the genes for both RNR subunits, R1 and R2, was high in proliferating cells, but was markedly down-regulated in parallel with adipocyte differentiation. NE stimulation, which promotes DNA synthesis and proliferation of brown preadipocytes, resulted in an increased expression of the R2 gene in proliferating cells (1.6-fold), but was without effect on R1 expression. In contrast, NE stimulation of confluent differentiating brown adipocytes reduced both R1 and R2 expression. The NE stimulation of R2 expression in preadipocytes was mimicked by forskolin and abolished by H89, demonstrating mediation via cAMP and protein kinase A (PKA). Also, inhibitors of Src and of Erk1/2 kinases markedly reduced NE-stimulated R2 expression. We conclude that adrenergic stimulation of brown adipocytes by NE specifically elevates expression of the RNR subunit R2 gene in the proliferative stage of brown adipocyte development, the mediating pathway being a cAMP/PKA cascade further involving Src and the MAP kinase Erk1/2. These results suggest that adrenergic stimulation of brown adipocyte proliferation may act at the level of gene expression of the limiting subunit for RNR activity, R2, and demonstrate a qualitative switch in the response of the R2 gene to cAMP-elevating agents as a consequence of the switch from proliferating to differentiating cell status.

Distinct Transcriptional Profiles of Adipogenesisin Vivo and in Vitro

Obesity, defined as an increase in adipose tissue mass, is the most prevalent nutritional disorder in industrialized countries and is a growing problem in developing countries. An increase in adipose tissue mass can be the result of the production of new fat cells through the process of adipogenesis and/or the deposition of increased amounts of cytoplasmic triglyceride per cell. Although much has been learned about the differentiation of adipocytes in vitro, less is known about the molecular basis for the mechanisms regulating adipogenesis in vivo. Here oligonucleotide microarrays have been used to compare the patterns of gene expression in preadipocytes and adipocytes in vitro and in vivo. These data indicate that the cellular programs associated with adipocyte differentiation are considerably more complex than previously appreciated and that a greater number of heretofore uncharacterized gene regulatory events are activated during this process in vitro. In addition, the gene expression changes associated with adipocyte development in vivo and in vitro, while overlapping, are in some respects quite different. These data further suggest that one or more transcriptional programs are activated exclusively in vivo to generate the full adipocyte phenotype. This gene expression survey now sets the stage for further studies to dissect the molecular differences between in vivo and in vitro adipocytes.

Opposite Effects of Androgens and Estrogens on Adipogenesis in Rat Preadipocytes: Evidence for Sex and Site-Related Specificities and Possible Involvement of Insulin-Like Growth Factor 1 Receptor and Peroxisome Proliferator-Activated Receptor 2

To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17β-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (×2) of 17β-estradiol (0.01μ m) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 μm) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17β-estradiol (0.01μ m), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariectomized female rats, 17β-estradiol significantly increased (×1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor γ2 was up-regulated by 17β-estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 β-estradiol (0.01 μm) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 μm) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor γ2 expression (estrogens).

Stimulation of adipose differentiation related protein (ADRP) expression in adipocyte precursors by long-chain fatty acids.

Adipose differentiation related protein (ADRP) is a 50-kDa protein expressed in adipocytes and transcriptionally activated when adipocyte precursors differentiate into mature adipocytes. Recent experiments have demonstrated that ADRP is a fatty acid binding protein that specifically facilitates the uptake of long-chain fatty acids. The present investigation provides evidence that ADRP mRNA and protein expression in preadipocytes is stimulated by fatty acids in a time- and dose-dependent fashion. ADRP mRNA expression was maximally stimulated at fatty acid concentrations of or above 10(-5) M. Stimulation of ADRP expression was observed with the nonmetabolizable fatty acid 2-bromopalmitate and with natural fatty acids. Stimulation of ADRP mRNA expression by fatty acids peaked between 5 and 8 hr and decreased by 24 hr. Stimulation of ADRP expression by fatty acids was completely inhibited by treatment with actinomycin D, suggesting that fatty acid stimulates ADRP gene expression at the transcriptional level. Comparison of the effect of several fatty acids with varying carbon chain lengths indicated that long-chain fatty acids were active in stimulating ADRP, whereas short-chain fatty acids such as caproate and 2-bromooctanoate had no effect. The degree of saturation of fatty acids did not influence their ability to stimulate ADRP expression. These studies provide new information on the regulation of ADRP and identify a new target regulated by fatty acids during adipose differentiation.

Identification of a Functional Peroxisome Proliferator-responsive Element in the Murine Fatty Acid Transport Protein Gene

Fatty acid transport protein (FATP), a plasma membrane protein implicated in controlling adipocyte transmembrane fatty acid flux, is up-regulated as a consequence of adipocyte differentiation and down-regulated by insulin. Based upon the sequence of the FATP gene upstream region (Hui, T. Y., Frohnert, B. I., Smith, A. J., Schaffer, J. A., and Bernlohr, D. A. (1998) J. Biol. Chem. 273, 27420-27429) a putative peroxisome proliferator-activated receptor response element (PPRE) is present from -458 to -474. To determine whether the FATP PPRE was functional, and responded to lipid activators, transient transfection of FATP-luciferase reporter constructs into CV-1 and 3T3-L1 cells was carried out. In CV-1 cells, FATP-luciferase activity was up-regulated 4- and 5.5-fold, respectively, by PPARalpha and PPARgamma in the presence of their respective activators in a PPRE-dependent mechanism. PPARdelta, however, was unable to mediate transcriptional activation under any condition. In 3T3-L1 cells, the PPRE conferred a small but significant increase in expression in preadipocytes, as well as a more robust up-regulation of FATP expression in adipocytes. Furthermore, the PPRE conferred the ability for luciferase expression to be up-regulated by activators of both PPARgamma and retinoid X receptor alpha (RXRalpha) in a synergistic manner. PPARalpha and PPARdelta activators did not up-regulate FATP expression in 3T3-L1 adipocytes, however, suggesting that these two subtypes do not play a significant role in differentiation-dependent activation in fat cells. Electromobility shift assays showed that all three PPAR subtypes were able to bind specifically to the PPRE as heterodimers with RXRalpha. Nuclear extracts from 3T3-L1 adipocytes also showed a specific gel-shift complex with the FATP PPRE. To correlate the expression of FATP to its physiological function, treatment of 3T3-L1 adipocytes with PPARgamma and RXRalpha activators resulted in an increased uptake of oleate. Moreover, linoleic acid, a physiological ligand, up-regulated FATP expression 2-fold in a PPRE-dependent manner. These results demonstrate that the FATP gene possesses a functional PPRE and is up-regulated by activators of PPARalpha and PPARgamma, thereby linking the activity of the protein to the expression of its gene. Moreover, these results have implications for the mechanism by which certain PPARgamma activators such as the antidiabetic thiazolidinedione drugs affect adipose lipid metabolism.

Long chain fatty acids as modulators of gene transcription in preadipose cells.

During the last years, it has been clearly established that long-chain fatty acids act as modulators of gene expression in various tissues, such as adipose tissue, intestine and liver. This transcriptional action of fatty acids explains in part adaptation mechanisms of tissues to nutritional changes and especially to high-fat diets by increasing expression of proteins involved in lipid catabolism in liver and fatty acid uptake and utilization in other tissues. It is now clearly demonstrated that some of these transcriptional effects of fatty acids are mediated by activation of specific nuclear hormone receptors, called peroxisome proliferator-activated receptors (PPARs). These findings will be discussed with a special reference to control of gene expression in preadipocytes and adipose tissue development.

CAMP Activates the Expression of Stearoyl-CoA Desaturase Gene 1 during Early Preadipocyte Differentiation

Stearoyl-CoA desaturase gene 1 (scd1) mRNA levels were induced during the 1st day of 3T3-L1 differentiation. scd1 expression had previously been observed only in late-stage differentiation, during the period of triacylglycerol accumulation. The induction of 3T3-L1 differentiation requires treatment with insulin, dexamethasone, and an agent that increases intracellular cAMP levels. Treatment of preadipocytes with cAMP-elevating agents caused an increase in scd1 mRNA concentrations. Insulin and dexamethasone had no effect on scd1 mRNA expression in preadipocytes. The increase in mRNA resulted from transcriptional activation of the scd1 gene. 8-Bromo-cAMP treatment of differentiated adipocytes had no effect on scd1 mRNA levels, suggesting a preadipocyte-specific effect. The increase in scd1 mRNA occurred maximally after 6 h of cAMP treatment and was shown to require protein synthesis. Deletion and mutagenesis analyses have localized the cAMP response to sequence within the first 250 base pairs of the 5'-flanking region of the scd1 gene. Treatment with phorbol ester enhanced the induction of scd1 mRNA by cAMP, suggesting the involvement of AP-2 as a mediator of the cAMP response. The induction of scd1 expression by cAMP during early differentiation was distinct from that observed during late adipocyte development. This early expression presents a novel regulated function of scd1 during the differentiation process, independent of its lipogenic role in late adipocyte development. The induction of scd1 also introduces a role for cAMP in 3T3-L1 differentiation.

Retinoic acid and dexamethasone interact to regulate S14 gene transcription in 3T3-F442A adipocytes

We have examined the effects of retinoic acid (RA) on S14 gene expression in 3T3-F442A preadipocytes and adipocytes. RA induced mRNA s14 14-fold in adipocytes, but had no effect on S14 gene expression in preadipocytes. Northern analysis of retinoic acid receptor (RAR) isoforms revealed the presence of RARα, but not RARβ, in both preadipocytes and adipocytes. These results suggest that adipocytespecific factors expressed during differentiation may be required for RA control of S14 gene expression in cultured adipocytes. When compared to dexamethasone (DEX), RA was a weak inducer of S14 gene expression. However, when added together, RA and DEX acted synergistically to induce S14 gene expression. Since changes in S14 gene transcription paralleled changes in mRNA s14 levels, the principal target for RA and DEX action on S14 gene expression was at the level of gene transcription. In the presence of DEX (0.1 nM), RA effects on S14 gene expression were dose-dependent (ED 50 = 5 nM) and rapid (within 4 h). In contrast to S14, RA inhibited glycerol 3-phosphate dehydrogenase (GPD) gene transcription and mRNA GDP abundance by 75%. The effects of RA on S14 and GPD gene transcription in cultured adipocytes suggest that RA action may extend beyond growth and differentiation to include effects on lipid metabolism.

Control of the adipsin gene in adipocyte differentiation. Identification of distinct nuclear factors binding to single- and double-stranded DNA.

The mouse adipsin gene encodes a serine protease with complement factor D activity that is expressed during adipocyte differentiation and is deficient in several animal models of obesity. We have investigated the regulation of adipsin expression by transfecting preadipocytes and adipocytes with plasmids containing the 5'-flanking region of the adipsin gene linked to a reporter gene. Constructions containing a -950 to +35 segment of the adipsin promoter were preferentially expressed in adipose cells. Deletion experiments identified a region from -114 to -38 which contains a large inverted repeat sequence and negatively regulated gene expression in preadipocytes and positively regulated expression in fat cells. Exonuclease III protection and gel retardation assays indicated that this region of duplex DNA had multiple binding sites for nuclear factors, several of which were preadipose specific. In addition, we also identified two distinct factors that bound symmetrically and sequence specifically to the inverted repeat sequences only when they were in single-stranded form; one of these factors was induced during adipocyte differentiation. These results suggest that the control of the adipsin promoter in differentiation may involve an interplay of multiple regulated DNA-binding proteins, including two that have preferential affinity for single-stranded DNA.