C/EBPα is indispensable for PML/RARα-mediated suppression of long non-coding RNA NEAT1 in acute promyelocytic leukemia cells.

Better understanding of the transcriptional regulatory network in acute promyelocytic leukemia (APL) cells is critical to illustrate the pathogenesis of other types of acute myeloid leukemia. Previous studies have primarily focused on the retinoic acid signaling pathway and how it is interfered with by promyelocytic leukemia/retinoic acid receptor-α (PML/RARα) fusion protein. However, this hardly explains how APL cells are blocked at the promyelocytic stage. Here, we demonstrated that C/EBPα bound and transactivated the promoter of long non-coding RNA NEAT1, an essential element for terminal differentiation of APL cells, through C/EBP binding sites. More importantly, PML/RARα repressed C/EBPα-mediated transactivation of NEAT1 through binding to NEAT1 promoter. Consistently, mutation of the C/EBP sites or deletion of retinoic acid responsive elements (RAREs) and RARE half motifs abrogated the PML/RARα-mediated repression. Moreover, silencing of C/EBPα attenuated ATRA-induced NEAT1 upregulation and APL cell differentiation. Finally, simultaneous knockdown of C/EBPα and C/EBPß reduces ATRA-induced upregulation of C/EBPε and dramatically impaired NEAT1 activation and APL cell differentiation. In sum, C/EBPα binds and transactivates NEAT1 whereas PML/RARα represses this process. This study describes an essential role for C/EBPα in PML/RARα-mediated repression of NEAT1 and suggests that PML/RARα could contribute to the pathogenesis of APL through suppressing C/EBPα targets.

Anti-oxidative and anti-adipogenic effects of caffeine in an in vitro model of Graves' orbitopathy.

Oxidative stress and adipogenesis play key roles in the pathogenesis of Graves' orbitopathy (GO). In this study, the therapeutic effects of caffeine on the reduction of oxidative stress and adipogenesis were evaluated in primary cultured GO orbital fibroblasts in vitro. Orbital fibroblasts were cultured from orbital connective tissues obtained from individuals with GO. Intracellular reactive oxygen species (ROS) levels induced by hydrogen peroxide or cigarette smoke extract and the expression of anti-oxidative enzymes were measured after caffeine treatment. After adipogenic differentiation and caffeine treatment, cells were stained with Oil Red O and the levels of peroxisome proliferator activator γ (PPARγ), C/EBPα, and C/EBPß were determined by western blot analysis. Hydrogen peroxide and cigarette smoke extract increased the levels of intracellular ROS and anti-oxidative enzymes, which decreased in a dose-dependent manner upon pretreatment with caffeine in GO orbital fibroblasts. Oil Red-O staining results revealed a decrease in lipid droplets; furthermore, PPARγ, C/EBPα, and C/EBPß protein expression levels were inhibited upon treatment with caffeine during adipocyte differentiation. In conclusion, caffeine decreased oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. These findings may contribute to the development of new types of caffeine-containing pharmacological agents for use in the management of GO.

Ethanol extracts of Aster yomena (Kitam.) Honda inhibit adipogenesis through the activation of the AMPK signaling pathway in 3T3-L1 preadipocytes.

The leaves of Aster yomena (Kitam.) Honda have long been used as a traditional herb for treating disorders including coughs, asthma, and insect bites. According to recent studies, A. yomena leaf extracts have several pharmacological properties, including anti-inflammatory, antioxidant, and anti-asthmatic activities. However, little information is available regarding their anti-obesity effect. In this study, we investigated the inhibitory effect of the ethanol extracts of A. yomena leaves (EEAY) on adipocyte differentiation and adipogenesis using 3T3-L1 preadipocytes. When 3T3-L1 preadipocytes were treated with various concentrations of EEAY (ranging from non-toxic), the number of lipid droplets, lipid content, and triglyceride production, the typical characteristics of adipocytes, were suppressed in a concentration-dependent manner. During this process, EEAY significantly reduced the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein α and ß, and sterol regulatory element-binding protein-1c. In addition, EEAY was also found to potently inhibit the expression of adipocyte-specific genes, including adipocyte fatty acid-binding protein and leptin. In particular, EEAY treatment effectively enhanced the activation of the AMP-activated protein kinase (AMPK) signaling pathway; however, the co-treatment with compound C, an inhibitor of AMPK, significantly restored the EEAY-induced inhibition of pro-adipogenic transcription factors and adipocyte-specific genes. These results indicate that EEAY may exert an anti-obesity effect by controlling the AMPK signaling pathway, suggesting that the leaf extract of A. yomena may be a potential anti-obesity agent.

MicroRNA-124 inhibits the progression of adjuvant-induced arthritis in rats.

OBJECTIVE: MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. METHODS: AIA was induced in Lewis rats by injecting incomplete Freund's adjuvant with heat-killed Mycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and real-time RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining. RESULTS: We found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor κB ligand (RANKL), integrin ß1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3'UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein α mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3'-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. CONCLUSIONS: We found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis.

Rutin Stimulates Adipocyte Differentiation and Adiponectin Secretion in 3T3-L1 Adipocytes.

Rutin is aflavonoid, which is found in many plants. It has been shown to reduce blood glucose and increase insulin levels in diabetic rats. In the present study, the authors aimed to elucidate the molecular basis for the observed antidiabetic activity using murine 3T3-L1 preadipocyte cultures. The treatment of differentiating 3T3-L1 cells with rutin at concentrations of 3, 10, 30 and 100 µM significantly increased lipid accumulation and mRNA expression of transcription factors, such as peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein alpha, and adipocyte fatty acid-binding protein. Furthermore, rutin at concentrations of 10, 30 and 100 µM increased adiponectin mRNA expression together with stimulating the secretion of adiponectin in differentiating 3T3-L1 cells. These results indicate that the stimulatory effect of rutin on adipocyte differentiation likely occurs through up-regulation of adipogenic transcription factors and downstream adipocyte-specific gene expression. Such effects of rutin on adiponectin secretion and adipocyte activity may account for, at least in part, the antidiabetic effects of consumption of food containing rutin.

The edible red alga, Gracilaria verrucosa, inhibits lipid accumulation and ROS production, but improves glucose uptake in 3T3-L1 cells.

Gracilaria verrucosa is a red alga that is widely distributed in seaside areas of many countries. We examined the effect of G. verrucosa extract on adipogenesis, reactive oxygen species (ROS) production, and glucose uptake in 3T3-L1 cells. Oil red O staining and a nitroblue tetrazolium assay showed that G. verrucosa extract inhibited lipid accumulation and ROS production, respectively. mRNA levels of adipogenic transcription factors, peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha, as well as of their target gene, adipocyte protein 2, were reduced upon treatment with G. verrucosa extract. However, G. verrucosa extract increased glucose uptake, glucose transporter-4 expression, and AMP-activated protein kinaseα (AMPKα) phosphorylation compared to the control. Our results suggest that the anti-adipogenic and insulin-sensitive effects of G. verrucosa extract can be recapitulated to activation of AMPKα.

Resveratrol inhibits cell differentiation in 3T3-L1 adipocytes via activation of AMPK.

Resveratrol (Res) is a natural polyphenolic compound with anti-inflammatory and antioxidant properties. Also, Res can inhibit lipogenesis and adipocyte differentiation. However, the underlying mechanisms of Res's functions remain largely unknown. AMP-activated protein kinase (AMPK) is a key player in adipocyte differentiation. Therefore, the purpose of our study was to determine the role played by AMPK in the Res-mediated regulation of adipocyte differentiation. Incubation of 3T3-L1 cells with Res confirmed that Res inhibited adipocyte differentiation. The phosphorylation of AMPKα was increased by Res in a dose-dependent manner, while total AMPKα levels were unchanged, and peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c) levels were decreased. Interestingly, pretreatment with AMPKα siRNA and Res promoted adipocyte differentiation, while the decrease of p-AMPKα increased PPARγ, C/EBPα, and SREBP-1c protein expression. Our study shows that Res is capable of inhibiting lipogenesis and differentiation of 3T3-L1 adipocytes via activation of AMPK, suggesting its potential therapeutic application in the treatment or prevention of obesity.

Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells.

Arsenic trioxide (ATO) as an anti-tumor drug could induce differentiation and apoptosis in tumor cells. Mesenchymal stem cells (MSCs) play important roles in the hematogenesis of bone marrow. Many reports have shown that the disorder of MSC adipogenic and osteogenic differentiation occurs in some diseases. However, reports about the effects of ATO on MSCs are limited. In this study, we found that 1 µM ATO promoted MSC senescence mainly through p21, although it had no effect on apoptosis at this dose. Furthermore, ATO promoted adipogenic differentiation, but inhibited osteogenic differentiation in MSCs. Our study also showed that CCAAT/enhancer-binding protein alpha C/EBPα and peroxisome proliferator-activated receptor gamma PPARγ might be involved in the regulation of adipogenic and osteogenic differentiation induced by ATO. Our results indicated that ATO may exert an anti-tumor effect by influencing bone marrow micro-environment. Moreover, it may regulate the adipogenic and osteogenic differentiation of MSCs.

Triptolide inhibits IL-12/IL-23 expression in APCs via CCAAT/enhancer-binding protein alpha.

Triptolide is a biologically active component purified from Chinese herbal plant Tripterygium wilfordii Hook F. It is widely used in East Asia for treatment of systemic lupus erythematosus, rheumatoid arthritis, nephritis, Bechect's disease, psoriasis, atopic dermatitis, and asthma. However, its immunological mechanisms are poorly understood. IL-12 and IL-23 are closely related heterodimeric cytokines that share the common subunit p40. They are produced by APCs and are key factors in the generation and effector functions of Th1 and Th17 cells, respectively. They have been strongly implicated in the pathogenesis of several autoimmune disorders. In this study, we investigated the molecular mechanism whereby triptolide inhibits the expression of the p40 gene in APCs. We demonstrate that triptolide does so at the transcriptional level in part through targeting CCAAT/enhancer-binding protein-alpha (C/EBPalpha), which directly interacts with the p40 promoter and inhibits its transcription in inflammatory macrophages. Triptolide can activate the transcription of C/EBPalpha, and phosphorylation of Ser21 and Thr222/226 critical for C/EBPalpha inhibition of p40. Further, activation of C/EBPalpha by triptolide is dependent on upstream kinases ERK1/2 and Akt-GSK3beta. This study provides mechanistic insights into the immunomodulatory capacity of triptolide and has strong implications for its therapeutic applications in autoimmune diseases.

Adipose tissue fat accumulation is reduced by a single intraperitoneal injection of peroxisome proliferator-activated receptor gamma agonist when given to newly hatched chicks.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a transcription factor that regulates adipocyte differentiation and modulates lipid metabolism in mammals. The aim of the present study was to investigate whether the administration of PPARgamma ligands, adipogenic cocktail, or both to newly hatched chicks regulates adipocyte differentiation in vivo and modulates fat deposition in growing broiler chickens. Levels of PPARgamma, CCAAT/enhancer binding protein alpha, and adipocyte fatty acid-binding protein mRNA in the abdominal fat pad of 7-d-old broiler chicks given a single intraperitoneal dose of troglitazone, a synthetic PPARgamma ligand, at 1 d old were significantly greater than those in control chickens. This suggests administration of troglitazone enhanced adipocyte differentiation in vivo. Adipose tissue weight in 28-d-old chickens similarly administered triolein emulsion containing troglitazone or adipogenic cocktail (i.e., dexamethasone, insulin, isobutyl-methylxanthine, and oleic acid) was also significantly less than that of control chickens. However, there was no significant difference in BW between treated and control chickens. Although BW and carcass composition were not different between troglitazone-treated and control chickens, at 48 d of age abdominal fat pad weight and feed intake were significantly decreased in chickens treated with troglitazone compared with controls. These results demonstrate that a single intraperitoneal injection of troglitazone to newly hatched chicks reduces fat deposition in mature broiler chickens.

Involvement of SREBPs in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced disruption of lipid metabolism in male guinea pig.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has multiple toxic effects causing a wasting syndrome characterized by a loss of body weight accompanied by a decrease in adipose tissue weight. To elucidate the mechanism behind this syndrome, we investigated the changes in lipid metabolism 7 and 21 days after a single intraperitoneal injection of TCDD at 1 microg/kg body weight to male guinea pigs. TCDD caused the symptoms of the syndrome, body weight loss with a decrease in adipose tissue weight, while it increased the levels of triacylglycerols, total cholesterols, and free fatty acids in plasma. On day 7, TCDD decreased the levels of CCAAT/enhancer binding protein (C/EBP) alpha, peroxisome proliferator activated receptor gamma, and glucose transporter 4, adipogenesis-related factors, in adipose tissue, whereas the levels of retinoid X receptor alpha, C/EBPbeta, C/EBPdelta, and c-Myc remained unchanged. TCDD also reduced the levels of both p125 precursor and p68 active forms of sterol regulatory element binding protein (SREBP)-1 and -2, the lipogenesis-related factors, and downregulated their DNA binding activity in adipose tissue, while it raised the levels of their p68 active forms and increased their DNA binding activity in the liver. TCDD decreased mRNA and protein levels of acetyl-CoA carboxylase and HMG-CoA synthase in the liver and adipose tissue. Similar results were obtained on day 21. These results suggest that TCDD disrupts lipid metabolism through changes in the expression levels of the adipogenesis-related and lipogenesis-related proteins in the liver and adipose tissue, and SREBPs would be involved in the development of the wasting syndrome.

CCAAT/enhancer binding protein-alpha is down-regulated by toll-like receptor agonists in microglial cells.

The transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha) can regulate the expression of important genes in the inflammatory response, but little is known about its role in glial activation. By using primary cortical murine glial cultures, we show that C/EBPalpha is expressed by microglial cells in vitro. Lipopolysaccharide (LPS) down-regulates C/EBPalpha mRNA at 2 hr and all C/EBPalpha protein isoforms at 4 hr. This effect is elicited by LPS concentrations >/=100 pg/ml. LPS-induced C/EBPalpha down-regulation occurs in microglial cells both in mixed glial and in microglial-enriched cultures. As seen with LPS, other toll-like receptor agonists (polyinosinic-polycytidylic acid, peptidoglycan from Staphylococcus aureus, and the oligonucleotide CpG1668) also down-regulate C/EBPalpha whereas cytokines such as interleukin-1beta, interleukin-6, macrophage-colony stimulating factor, and interferon-gamma do not. These findings suggest that C/EBPalpha down-regulation in activated microglia could play an important role in the increased expression of genes that are potentially pathogenic in a variety of neurological disorders.

Regulation of adipocyte differentiation by PEGylated all-trans retinoic acid: reduced cytotoxicity and attenuated lipid accumulation.

Obesity is major risk factor for many disorders, including diabetes, hypertension and heart disease. Unfortunately, there is a dearth of therapeutic agents available to clinicians for the treatment of obesity. The principal aim of this study was to investigate whether PEGylated all-trans retinoic acid (PRA) can have favorable stability and biological activity in 3T3-L1 preadipocytes as an antiobesity drug. Here, we found that PRA inhibits the process of adipogenesis, including survival of adipocytes and differentiation to mature adipocytes. The results showed that RA nanoparticles (NPs) were prepared by PEGylation; below 200 nm, PRA-NPs were obtained. Moreover, PRA decreased glycerol-3-phosphate dehydrogenase activity in 3T3-L1 preadipocytes by acting with major adipocyte marker proteins such as PPARgamma2, C/EBPalpha and aP2 modulators. Apoptosis, in addition, increased as the level of RA increased from 10 to 20 microM, whereas PRA reduced apoptosis with increasing concentrations. Our data suggest that PRA-NP has potential as an antiobesity drug carrier due to its small particle size and PEGylated core-shell structure. In addition, our results suggest that PRA inhibits the process of adipogenesis and may be developed to treat obesity. Based on these results, PRA is suitable for adipocyte studies, and an enhanced effect of PRA with adipocyte differentiation offers a challenging approach for pharmaceutical applications.

Berberine inhibits 3T3-L1 adipocyte differentiation through the PPARgamma pathway.

Berberine (BBR), a compound purified from Cortidis rhizoma, reduces serum cholesterol, triglycerides, and LDL-cholesterol of hypercholesterolemic patients and high fat diet fed animals, and increases hepatic LDLR mRNA and protein levels through a post-transcriptional mechanism. BBR also enhances the hypoglycemic action of insulin in diabetic animal models. Here, we show that BBR inhibits the differentiation of 3T3-L1 preadipocytes induced by DM and suppresses the mitotic clonal expansion of 3T3-L1 preadipocytes in a time- and dose-dependent manner. Gene expression analysis and Western blot analysis reveal that the BBR inhibits the mRNA and protein levels of adipogenesis related transcription factors PPARgamma and C/EBPalpha and their upstream regulator, C/EBPbeta. Reporter gene assays demonstrate that the full-length PPARgamma and alpha transcription activities are inhibited by BBR. Using real-time PCR, we have also found that the PPAR target genes that are involved in adipocyte differentiation, such as aP2, CD36, ACO, LPL, and other adipocyte markers, are suppressed by BBR. These studies suggest that BBR works on multiple molecular targets as an inhibitor of PPARgamma and alpha, and is a potential weight reducing, hypolipidemic, and hypoglycemic drug.

A novel retinoic/butyric hyaluronan ester for the treatment of acute promyelocytic leukemia: preliminary preclinical results.

All-trans retinoic acid (ATRA) represents the therapy of choice for patients with acute promyelocytic leukemia (APL). However, patients often relapse due to ATRA-resistance. The molecular basis of APL alterations indicates that addition of a histone deacetylase inhibitor to ATRA may restore the sensitivity to retinoids. We explored the in vitro and in vivo effects of a novel retinoic/butyric hyaluronan ester (HBR) on a retinoic acid (RA)-sensitive human myeloid cell line, NB4, and on its RA-resistant subclone, NB4.007/6. In vitro, HBR induced growth arrest and terminal differentiation in RA-sensitive NB4 cells (as confirmed by an increased expression of CD11 family members and nitroblue tetrazolium assay), whereas it inhibited the growth of RA-resistant cells by apoptosis, paralleled by an increase in the levels of caspase 3 and 7. In vivo, HBR treatment of NB4-inoculated severe combined immunodeficient mice resulted in a statistically significant increase in survival time (P<0.0001), comparable to that induced by a maximum tolerated dose of RA alone. Also on P388-inoculated mice, HBR was active in contrast to RA that was completely ineffective. Present findings suggest that, owing to the simultaneous presence of RA and an histone deacetylases inhibitor, HBR might be useful in controlling the proliferation of RA-resistant cells and the differentiation of RA-sensitive cells.

Suppression of adiponectin gene expression by histone deacetylase inhibitor valproic acid.

Valproic acid (VPA) has been used for the treatment of epilepsy and bipolar disorders for more than 30 yr. Obesity and insulin resistance are common side effects of VPA treatment. Adiponectin is an adipocyte-derived protein that plays an important role in controlling insulin sensitivity and glucose homeostasis. In this report, we examined the effects of VPA on adiponectin gene expression in C57BL/6J mice and in differentiated 3T3-L1 adipocytes. VPA treatment significantly decreased adiponectin protein and mRNA levels in both mice and 3T3-L1 adipocytes. The adipocyte study showed that VPA inhibited adiponectin gene expression in a dose- and time-dependent manner. Repression of adiponectin expression by VPA occurred at the transcription level and correlated with inhibition of histone deacetylase activity. Therapeutic concentrations of VPA increased overall histone acetylation and increased adiponectin promoter-driven luciferase expression in fibroblasts, but decreased adiponectin promoter activity in differentiated 3T3-L1 adipocytes. VPA treatment decreased adipogenic transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha) levels and binding of C/EBPalpha to the adiponectin promoter without altering the levels of peroxisome proliferator-activated receptor-gamma and steroid regulatory element binding protein-1. Furthermore, VPA did not suppress adiponectin gene expression in C/EBPalpha gene-deficient adipocytes that stably expressed exogenous peroxisome proliferator-activated receptor-gamma2. Together, these results demonstrate that histone deacetylase inhibitor VPA suppresses adiponectin gene expression in mature adipocytes. The study also provides evidence that diminished C/EBPalpha protein level and decreased binding at the adiponectin promoter mediate the inhibitory effects of VPA on adiponectin gene transcription.

Isomer-specific regulation of differentiating pig preadipocytes by conjugated linoleic acids.

Conjugated linoleic acids are a group of geometric and positional isomers of linoleic acid that decrease body fat in growing animals by a poorly understood mechanism. The objective of this study was to investigate the isomer-specific effect of CLA on the proliferation and differentiation of pig preadipocytes in primary culture. The effect of CLA on preadipocyte proliferation was determined using cleavage of the tetrazolium salt, WST-1, as a marker for proliferation. Preadipocyte number was decreased in a dose-dependent fashion by trans-12,cis-10 CLA (P < 0.05). No other fatty acid affected preadipocyte number. Differentiation was monitored on d 10 after induction morphologically, enzymatically, and by measuring the mRNA abundance of key adipogenic transcription factors. Both a crude CLA preparation containing a mixture of CLA isomers (CLA-mix) and the pure trans-10,cis-12 CLA isomer inhibited glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent fashion, with trans-10,cis-12 CLA being more potent (P < 0.01) than the CLA-mix. Cis-9,trans-11 CLA failed to decrease GPDH activity; however, increasing concentrations of cis-9,trans-11 CLA tended to blunt the inhibitory effect of trans-10,cis-12 CLA on GPDH activity (P < 0.09), suggesting that cis-9,trans-11 CLA may antagonize the action of trans-10,cis-12 CLA in porcine adipocytes. Finally, the isomer-specific effect of CLA on adipogenic transcription factor gene expression was investigated. Trans-10,cis-12 CLA decreased expression of peroxisome proliferator-activated receptor gamma (PPAR gamma; P < 0.01) and sterol regulatory element-binding protein-1c (SREBP-1c; P < 0.05) mRNA, while failing to alter the expression of CCAAT/enhancer binding protein alpha (C/EBPalpha) mRNA. Interestingly, both the CLA-mix and the trans-10,cis-12 CLA isomer increased the mRNA abundance of chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF; P < 0.002). No other fatty acid affected COUP-TF mRNA levels. Collectively these data support the concept that CLA decreases fat accretion in pigs, in part by inhibiting preadipocyte proliferation and differentiation, with trans-10,cis-12 CLA being an active isomer eliciting these effects. Furthermore, trans-10,cis-12 CLA inhibits porcine preadipocyte differentiation by a mechanism that involves the down-regulation of PPARgamma and SREBP-1c mRNA. This mechanism is independent of changes in C/EBPalpha mRNA abundance and may involve COUP-TF.

Differentiation of pluripotent C3H10T1/2 cells rapidly elevates CYP1B1 through a novel process that overcomes a loss of Ah Receptor.

Stimulation of C3H10T1/2 cells by an adipogenic hormonal mixture (IDM) consisting of insulin (I), dexamethasone (D), and methylisobutylxanthine (M) substantially induces cytochrome P450 (CYP) 1B1 expression. This stimulation represents up to 40% of the level produced by maximum activation of the arylhydrocarbon receptor (AhR) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dexamethasone and methylisobutylxanthine in combination produced near maximum elevation of CYP1B1 along with a subsequent decline in AhR that paralleled the rise in peroxisome proliferator-activated receptorgamma1 (PPARgamma1). Inhibitors of AhR activity, which block TCDD induction, did not affect this increase of CYP1B1 expression, which was, therefore, independent of AhR activity. These responses were unaffected by inhibition of DNA synthesis, which was required for PPARgamma1 induction and terminal differentiation. Induction of CYP1B1 mRNA was paralleled by increased CYP1B1 promoter-luciferase reporter activity. The initial 0.8kb of promoter region, which was sufficient for 24h near maximum stimulation, did not contain either the key AhR-responsive elements that mediate the TCDD response or CREB and SF1 elements that mediate cAMP stimulation of rat CYP1B1 in steroidogenic cells. This reporter response to IDM stimulation, but not to TCDD, was maintained in AhR-null fibroblasts. CYP1B1 expression, unlike TCDD induction, was stimulated by IDM in only about half the cells. CYP1B1 expression partially overlapped with PPARgamma expression, which was also inversely related in clonal sub-lines. CYP1B1 expression may, therefore, represent an early stage of differentiation that requires factors associated with DNA synthesis to subsequently generate PPARgamma1.

Inhibition of histone deacetylase activity by valproic acid blocks adipogenesis.

Adipogenesis is dependent on the sequential activation of transcription factors including the CCAAT/enhancer-binding proteins (C/EBP), peroxisome proliferator-activated receptor gamma (PPARgamma), and steroid regulatory element-binding protein (SREBP). We show that the mood stabilizing drug valproic acid (VPA; 0.5-2 mm) inhibits mouse 3T3 L1 and human preadipocyte differentiation, likely through its histone deacetylase (HDAC) inhibitory properties. The HDAC inhibitor trichostatin A (TSA) also inhibited adipogenesis, whereas the VPA analog valpromide, which does not possess HDAC inhibitory effects, did not prevent adipogenesis. Acute or chronic VPA treatment inhibited differentiation yet did not affect mitotic clonal expansion. VPA (1 mm) inhibited PPARgamma induced differentiation but does not activate a PPARgamma reporter gene, suggesting that it is not a PPARgamma ligand. VPA or TSA treatment reduced mRNA and protein levels of PPARgamma and SREBP1a. TSA reduced C/EBPalpha mRNA and protein levels, whereas VPA only produced a decrease in C/EBPalpha protein expression. Overall our results highlight a role for HDAC activity in adipogenesis that can be blocked by treatment with VPA.

Insulin-dependent adipogenesis in stromal ST2 cells derived from murine bone marrow.

ST2 cells, a cloned stromal-cell line from mouse bone marrow have the phenotypes of osteoblasts and hematopoietic supporting cells, but little is known about the adipogenesis in this cell line. We found that treatment of ST2 cells with a cocktail, a combination of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine (IBMX), induced adipocyte differentiation. The cells were adipocytic, as seen by the accumulation of triglycerides and with lipid droplets stained with Oil Red O. Expressions of the adipogenic transcriptional factors peroxisome-proliferator-activated receptor gamma and CCAAT-enhancer binding protein alpha were stimulated, triggering the expression of the late adipocyte-specific marker alpha-glycerophosphate-3-dehydrogenase. Unlike another bone marrow stromal cell line PA6, ST2 cells responded to the adipogenic effects of insulin, as do the extramedullary preadipocytes 3T3-L1. Like PA6 and 3T3-L1 cells, adipogenesis in ST2 cells was inhibited by 1,25-dihydroxyvitamin D3, retinoic acid, tumour necrosis factor alpha, and transforming growth factor beta. Therefore, ST2 cells can differentiate into osteoblasts, adipocytes, and hematopoietic supporting cells, in which the process of adipogenesis differs from that of bone marrow preadipocytes PA6, and resembles that of the extramedullary preadipocytes 3T3-L1.