Adipocyte Transcription Factors Research Articles

Inhibitory Effects of Ethyl Acetate-Soluble Fraction from Morus alba on Lipid Accumulation in 3T3-L1 Cells

Fruits of mulberry (Morus alba) have been widely used for therapeutic purposes in Asian countries for centuries. Treatment of 3T3-L1 cells with ethanolic extracts of M. alba decreased adipocyte differentiation at 100 microg/mL by 18.6%. Treatment suppressed mRNA levels of PPARgamma and C/EBPalpha expression in 3T3-L1 cells. However, the extract did not change free glycerol release from mature adipocytes. Thus, M. alba inhibited lipid accumulation by regulating transcription factors in 3T3-L1 adipocytes without a lipolytic effect. Among the soluble- fractions, the ethyl acetate-soluble fraction had the highest antiadipogenic effects on 3T3-L1 cells. This fraction decreasing intracellular lipid accumulation by 38.5% in response to treatment with 100 microg/mL. In addition, HPLC analysis of the ethyl acetate-soluble fraction of M. alba contained 167.7 microM of protocatechulic acid in 1 mg/mL of fraction, which inhibited lipid accumulation by 44.8% in response to treatment with 100 microM. From these results, M. alba is a possible candidate for regulating lipid accumulation in obesity.

Inhibitory effects of baicalin in the early stage of 3T3-L1 preadipocytes differentiation by down-regulation of PDK1/Akt phosphorylation

Baicalin is a flavonoid derived from the root of Scutellaria baicalensis and exhibits a broad spectrum of biological activities including anti-adipogenesis. However, the inhibitory role of baicalin in the early stage of 3T3-L1 adipocyte differentiation relevant to the signaling up-stream of peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding proteins (C/EBPs) expression is unclear, and is the subject of the present investigation. We used 3T3-L1 preadipocytes for adipocyte differentiation, Oil Red-O staining for the intracellular lipid accumulation assay, and real-time polymerase chain reaction (RT-PCR) for assaying the expression of major adipocyte transcription factors. We found that baicalin markedly suppressed the Akt phosphorylation in early stage of adipocytes differentiation. In addition, we observed that baicalin and LY294002 (as an inhibitor of Akt phosphorylation) significantly inhibited adipocyte differentiation by down-regulating several adipocyte-specific transcription factors, including PPAR-γ and C/EBPs in 3T3-L1 preadipocytes. Furthermore, we observed that baicalin significantly suppressed the Akt phosphorylation by inhibiting phosphoinositide-dependent kinase 1 (PDK1). These results indicate that the anti-adipogenesis effect of baicalin involves down-regulation of major transcription factors in 3T3-L1 adipocyte differentiation including PPAR-γ, C/EBP-β, and C/EBP-α through the down-regulation of PDK1/Akt phosphorylation.

Acute Genome-Wide Effects of Rosiglitazone on PPARγ Transcriptional Networks in Adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation, and genome-wide studies indicate that it is involved in the induction of most adipocyte genes. Here we report, for the first time, the acute effects of the synthetic PPARγ agonist rosiglitazone on the transcriptional network of PPARγ in adipocytes. Treatment with rosiglitazone for 1 hour leads to acute transcriptional activation as well as repression of a number of genes as determined by genome-wide RNA polymerase II occupancy. Unlike what has been shown for many other nuclear receptors, agonist treatment does not lead to major changes in the occurrence of PPARγ binding sites. However, rosiglitazone promotes PPARγ occupancy at many preexisting sites, and this is paralleled by increased occupancy of the mediator subunit MED1. The increase in PPARγ and MED1 binding is correlated with an increase in transcription of nearby genes, indicating that rosiglitazone, in addition to activating the receptor, also promotes its association with DNA, and that this is causally linked to recruitment of mediator and activation of genes. Notably, both rosiglitazone-activated and -repressed genes are induced during adipogenesis. However, rosiglitazone-activated genes are markedly more associated with PPARγ than repressed genes and are highly dependent on PPARγ for expression in adipocytes. By contrast, repressed genes are associated with the other key adipocyte transcription factor CCAAT-enhancer binding proteinα (C/EBPα), and their expression is more dependent on C/EBPα. This suggests that the relative occupancies of PPARγ and C/EBPα are critical for whether genes will be induced or repressed by PPARγ agonist.

Red yeast rice‐garlic inhibits the markers of differentiation in 3T3‐L1 preadipocytes

AbstractThis study investigates the hypothesis that the ethyl acetate extract of rice fermented with red yeast (Monascuspilosus) in medium containing garlic (RYRGE) inhibits the differentiation of 3T3‐L1 preadipocytes by down regulating cellular induction of PPARγ, and C/EBPα. Mouse 3T3‐L1 preadipocytes were treated with 0.0, 0.125, and 0.25 mg/mL of RYRGE, and Oil Red O staining, triglyceride accumulation and glyceradehyde‐3‐phosphate dehydrogenase (GPDH) activity were measured as markers of adipocyte differentiation. The protein and mRNA expressions of PPARγ and C/EBPα were measured using Western Blot and RT‐PCR, respectively. The mRNA expressions of leptin and aP2 genes were measured using RT‐PCR. RYRGE significantly inhibited lipid accumulation and GPDH activity in dose‐dependent manner (p<0.05). The mRNA and protein expressions of PPARγ and C/EBPα were dose‐dependently inhibited by RYRGE. Similarly, RYRGE decreased the gene expression of adipocyte fatty acid binding protein (aP2) and leptin in a dose‐dependent manner (p<0.05), which generally acts as an adipogenic marker proteins and C/EBPα and PPARγ target genes. These results indicate that RYRGE effectively attenuated adipogenesis by down‐regulating the expression of adipocyte transcription factors and related adipogenic gene expressions.

지방세포분화에서의 replication factor C 단백질의 발현조절

Adipocyte differentiation is an ordered multistep process requiring the sequential activation of several groups of adipogenic transcription factors, including CCAAT/enhancer-binding protein-α and peroxisome proliferator-activated receptor-γ, and coactivators. In previous reports, we identified that replication factor C 140 (RFC140) protein played a critical role in regulating adipocyte differentiation as a coactivator. Here, we show expressional regulation of RFC140 and small RFC subunit, RFC38, following characterization of gene promoter of RFC140 and RFC38. In addition, RFC140 increases PPARγ -mediated gene activation, resulting from direct protein-protein interaction of RFC140 and PPARγ. Taken together, these findings demonstrate that the regulated expression of RFC140 and RFC38 by specific adipocyte transcription factors is required for the adipocyte differentiation process.

Momordica charantia (bitter melon) inhibits primary human adipocyte differentiation by modulating adipogenic genes.

BackgroundEscalating trends of obesity and associated type 2 diabetes (T2D) has prompted an increase in the use of alternative and complementary functional foods. Momordica charantia or bitter melon (BM) that is traditionally used to treat diabetes and complications has been demonstrated to alleviate hyperglycemia as well as reduce adiposity in rodents. However, its effects on human adipocytes remain unknown. The objective of our study was to investigate the effects of BM juice (BMJ) on lipid accumulation and adipocyte differentiation transcription factors in primary human differentiating preadipocytes and adipocytes.MethodsCommercially available cryopreserved primary human preadipocytes were treated with and without BMJ during and after differentiation. Cytotoxicity, lipid accumulation, and adipogenic genes mRNA expression was measured by commercial enzymatic assay kits and semi-quantitative RT-PCR (RT-PCR).ResultsPreadipocytes treated with varying concentrations of BMJ during differentiation demonstrated significant reduction in lipid content with a concomitant reduction in mRNA expression of adipocyte transcription factors such as, peroxisome proliferator-associated receptor γ (PPARγ) and sterol regulatory element-binding protein 1c (SREBP-1c) and adipocytokine, resistin. Similarly, adipocytes treated with BMJ for 48 h demonstrated reduced lipid content, perilipin mRNA expression, and increased lipolysis as measured by the release of glycerol.ConclusionOur data suggests that BMJ is a potent inhibitor of lipogenesis and stimulator of lipolysis activity in human adipocytes. BMJ may therefore prove to be an effective complementary or alternative therapy to reduce adipogenesis in humans.

Peroxisome Proliferator-Activated Receptor γ Dances with Different Partners in Macrophage and Adipocytes

Peroxisome Proliferator-Activated Receptor γ Dances with Different Partners in Macrophage and Adipocytes

Comparative analysis of fatty acid-binding protein 4 promoters: Conservation of peroxisome proliferator-activated receptor binding sites1

The objective of this research was to further characterize the promoter regions of the bovine and porcine fatty acid-binding protein 4 (FABP4) genes relative to those of other mammals. The DNA sequences of FABP4 promoters for the human, mouse, cow, pig, and dog were obtained from the genomic database of the National Center for Biotechnology Information and also from the sequencing of bovine and porcine genomic DNA clones obtained by 5' PCR racing of genomic DNA. Sequence alignments of these FABP4 promoters using the basic local alignment search tool of the National Center for Biotechnology Information revealed 3 highly conserved promoter regions across the species. Two computational bioinformatics databases and the literature identified the conserved transcription factor-binding sites for C/EBP, adapter primer-1, and boxes of CAAT and TATA in the first conserved proximal promoter region, a direct repeat 1-type PPAR responsive element in the second distal conserved region, and another PPAR responsive element in the third distal conserved promoter region of FABP4 in all 5 mammals. Five new short interspersed repetitive elements (SINE) in the bovine FABP4 promoter and 2 new SINE in the porcine were found, but these SINE did not disrupt the 3 conserved regions, indicating that important regulatory elements are maintained regardless of evolutionary pressure. In conclusion, the conserved cis-acting elements, especially the 2 key adipocyte transcription factors C/EBP and PPAR, may contribute greatly to adipogenic regulation and adipose tissue-specific expression of FABP4 in these mammals. This helps to further characterize and decipher important cis-acting elements that are important for adipocyte development in adipose and muscle tissue.

SPARC Inhibits Adipogenesis by Its Enhancement of β-Catenin Signaling

SPARC (secreted protein acidic and rich in cysteine) modulates interactions between cells and extracellular matrix and is enriched in white adipose tissue. We have reported that SPARC-null mice accumulate significantly more fat than wild-type mice and maintain relatively high levels of serum leptin. We now show that SPARC inhibits adipogenesis in vitro. Specifically, recombinant SPARC inhibited (a) adipocyte differentiation of stromal-vascular cells isolated from murine white adipose tissue and (b) the expression of adipogenic transcription factors and adipocyte-specific genes. SPARC induced the accumulation and nuclear translocation of beta-catenin and subsequently enhanced the interaction of beta-catenin and T cell/lymphoid enhancer factor 1. The activity of integrin-linked kinase was required for the effect of SPARC on beta-catenin accumulation as well as extracellular matrix remodeling. During adipogenesis, fusiform preadipocytes change into sphere-shaped adipocytes and convert the extracellular matrix from a fibronectin-rich stroma to a laminin-rich basal lamina. SPARC retarded the morphological changes exhibited by preadipocytes during differentiation. In the presence of SPARC, the deposition of fibronectin was enhanced, and that of laminin was inhibited; in parallel, the expression of alpha5 integrin was enhanced, and that of alpha6 integrin was inhibited. Lithium chloride, which enhances the accumulation of beta-catenin, also inhibited the expression of alpha6 integrin. These findings demonstrate a role for SPARC in adipocyte morphogenesis and in signaling processes leading to terminal differentiation.

Adipose Inflammation, Insulin Resistance, and Cardiovascular Disease

Adiposity-associated inflammation and insulin resistance are strongly implicated in the development of type 2 diabetes and atherosclerotic cardiovascular disease. This article reviews the mechanisms of adipose inflammation, because these may represent therapeutic targets for insulin resistance and for prevention of metabolic and cardiovascular consequences of obesity. The initial insult in adipose inflammation and insulin resistance, mediated by macrophage recruitment and endogenous ligand activation of Toll-like receptors, is perpetuated through chemokine secretion, adipose retention of macrophages, and elaboration of pro-inflammatory adipocytokines. Activation of various kinases modulates adipocyte transcription factors, including peroxisome proliferator-activated receptor-gamma and NFkappaB, attenuating insulin signaling and increasing adipocytokine and free fatty acid secretion. Inflammation retards adipocyte differentiation and further exacerbates adipose dysfunction and inflammation. Paracrine and endocrine adipose inflammatory events induce a local and systemic inflammatory, insulin-resistant state promoting meta-bolic dyslipidemia, type 2 diabetes, and cardiovascular disease. Developing therapeutic strategies that target both adipose inflammation and insulin resistance may help to prevent type 2 diabetes and cardiovascular disease in the emerging epidemic of obesity.

Sequential Expression of Wnt/β-catenin Signal Pathway Related Genes and Adipocyte Transcription Factors During Porcine Adipose Tissue Development

To evaluate the influence of Wnt/β-catenin signal pathway on the porcine adipose tissue development and explore the mechanism, we detected the mRNA expression of Wnt/β-catenin signal pathway related genes: β-catenin, GSK3β, Fz1 and adipogenic transcription factors: PPARγ, C/EBPα and early differentiation marker gene LPL with semi-quantitative (SQ) RT-PCR method. Immunohistochemical method (IHC) was applied to qualitatively measure the sequential expression of β-catenin protein. The results of SQ RT-PCR showed that β-catenin highly expressed at the first day after birth, then decreased to a low plateau after 60 days, the expression of GSK3β and Fz1 also decreased with the development process of the porcine adipose tissue development. However, the sequential expression of PPARγ, C/EBPα, LPL appeared to be an opposite manner and kept at a high level after 60 days. The result of IHC showed that the expression of β-catenin protein was strong in nucleus and cytoplasm at the first day after birth, then tended to decline with the process of adipose tissue development and could be only found in cytoplasm after 30-day old. These results suggest that β-catenin plays an important role in the undifferentiated state maintenance of preadipocytes and the inhibition of porcine adipose tissue development, the mechanism maybe due to its regulation function on the adipogenic transcription factors PPARγ, C/EBPα and early differentiation marker gene LPL.

Transcriptional regulation of the proximal promoter of the murine pyruvate carboxylase gene in liver cells

Pyruvate carboxylase (PC) plays a crucial role in intermediary metabolism i.e. gluconeogenesis, lipogenesis and insulin secretion. Mammalian PC genes are regulated by 2 promoters, the proximal (P1) and the distal (P2) promoters which mediate alternative transcription in tissue‐specific manner. P1 promoter is active in liver and adipocytes while the P2 is active in pancreatic islets. In islets, P2 promoter is regulated by Sp1/Sp3, USF1, 2 and Foxa2 while P1 promoter is regulated by the PPARgamma. P1 is active in both liver and adipocytes, suggesting that this promoter is regulated by the distinct sets of liver and adipocyte transcription factors. To investigate this, we identify the liver‐specific transcription factors that regulate PC expression. We generate a series of 5’‐nested deletion mutants of the 2.3 kb P1 promoter of the mouse PC gene followed by transient transfection in HepG2 cells. This result showed the presence of 3 regulatory regions, located in regions A (−166/−80), B (−166/−256) and C (−335/−603). Region A contains 3 sites for Sp1/Sp3 while the region C contains a putative binding site for the liver‐enrich nuclear factor, HNF4alpha which could potentially bind to its responsive element identical to the PPRE. We are investigating the molecular interaction between HNF4 alpha to this site as well as identifying the nuclear proteins that bind region B of the P1 promoter.

TCDD suppresses insulin-responsive glucose transporter (GLUT-4) gene expression through C/EBP nuclear transcription factors in 3T3-L1 adipocytes

TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3-L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between -78 and -273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel-shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [(32)P]-labeled probe containing C/EBP response element equivalent of the -265 to -242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBPalpha and an increase in that of C/EBPbeta among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBPalpha, C/EBPbeta, or C/EBPdelta through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBPalpha or C/EBPbeta. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3-L1 cells.

Altered pre-lamin A processing is a common mechanism leading to lipodystrophy

Lipodystrophies are a heterogeneous group of human disorders characterized by the anomalous distribution of body fat associated with insulin resistance and altered lipid metabolism. The pathogenetic mechanism of inherited lipodystrophies is not yet clear; at the molecular level they have been linked to mutations of lamin A/C, peroxisome proliferator-activated receptor (PPARgamma) and other seemingly unrelated proteins. In this study, we examined lamin A/C processing in three laminopathies characterized by lipodystrophic phenotypes: Dunnigan type familial partial lipodystrophy, mandibuloacral dysplasia and atypical Werner's syndrome. We found that the lamin A precursor was specifically accumulated in lipodystrophy cells. Pre-lamin A was located at the nuclear envelope and co-localized with the adipocyte transcription factor sterol regulatory element binding protein 1 (SREBP1). Using co-immunoprecipitation experiments, we obtained the first demonstration of an in vivo interaction between SREBP1 and pre-lamin A. Binding of SREBP1 to the lamin A precursor was detected in patient fibroblasts as well as in control fibroblasts forced to accumulate pre-lamin A by farnesylation inhibitors. In contrast, SREBP1 did not interact in vivo with mature lamin A or C in cultured fibroblasts. To gain insights into the effect of pre-lamin A accumulation in adipose tissue, we inhibited lamin A precursor processing in 3T3-L1 pre-adipocytes. Our results show that pre-lamin A sequesters SREBP1 at the nuclear rim, thus decreasing the pool of active SREBP1 that normally activates PPARgamma and causing impairment of pre-adipocyte differentiation. This defect can be rescued by treatment with troglitazone, a known PPARgamma ligand activating the adipogenic program.

Interleukin 11 treatment alters the protein content of Gα i2 and adipogenic transcription factors in 3T3-L1 adipocytes

Exposure of fully differentiated 3T3-L1 adipocytes to 5 nM interleukin 11 (IL-11) resulted in an increase (1.9±0.5 fold) in the protein content for the heterotrimeric G protein Gα i2. This G protein has been suggested to be involved in the control of the insulin responsive glucose transporter (GLUT4) translocation to the plasma membrane. Conversely, IL-11 had no effect on the content of three other G proteins, involved in insulin action. The alteration in Gα i2 protein corresponds to and provides a molecular rationale for our previously described IL-11 induced increase in plasma membrane glucose transporter content and increased rate of glucose transport. In addition, treatment with the cytokine altered the protein content of several transcription factors, C/EBPα and CHOP-10 decreased while PPARγ and C/EBPβ increased. These changes in transcription factor content are consistent with an alteration of phenotype with the cells reverting to an earlier stage of the differentiation process in response to IL-11.

Plasticity and regulation of human bone marrow stromal osteoprogenitor cells: potential implication in the treatment of age-related bone loss.

Human bone marrow stroma contains pluripotent mesenchymal progenitor cells that can give rise to many mesenchymal lineages, including chondroblasts, adipocytes or osteoblasts. The differentiation of these cells towards a specific lineage is dependent on hormonal and local factors activating specific transcription factors. Attempts have been recently made to identify osteoprogenitor cells in the human bone marrow and to identify the molecular mechanisms responsible for lineage-specific differentiation of human bone marrow stromal cells. Using a clonal pluripotent human bone marrow stromal cell line with tri-potential characteristics, we have provided evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of human bone marrow stromal cells. We have also shown that administration of TGFss that regulates the expression of specific osteoblast and adipocyte transcription factors can promote osteoblast differentiation and inhibit adipocyte conversion of rat marrow stromal cells in vivo. This indicates that the reciprocal relationship between osteoblastogenesis and adipogenesis can be manipulated in vivo in order to improve bone formation. Future studies will have to identify key signals for lineage-specific differentiation of human marrow stromal cells. This may result in the development of therapeutic strategies to promote the differentiation of these cells towards the osteoblast lineage and to inhibit excessive bone marrow adipogenesis associated with aging.

Trans-10, cis-12 CLA inhibits differentiation of 3T3-L1 adipocytes and decreases PPARγ expression

The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) has been shown to reduce body fat gain in mice. However, the underlying molecular mechanism is not well characterized. Here we report evidence that trans-10, cis-12 (t10c12) CLA inhibits preadipocyte differentiation. Treating differentiating 3T3-L1 preadipocytes with t10c12 CLA and conjugated nonadecadienoic acid (CNA, a 19-carbon CLA cognate) resulted in decreased intracellular triglyceride accumulation and mRNA levels of the adipogenic gene fatty acid synthase and adipocyte lipid binding protein. T10c12 CLA and CNA also reduced protein levels of adipocyte transcription factors, peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α. Similarly, CLA reduced body fat gain and significantly inhibited the expression of PPARγ and its downstream target lipoprotein lipase in mouse adipose tissue. These observations indicate that CLA decreases body fat gain in part by inhibiting the differentiation of preadipocytes.

Transforming Growth Factor-β Inhibits Adipocyte Differentiation by Smad3 Interacting with CCAAT/Enhancer-binding Protein (C/EBP) and Repressing C/EBP Transactivation Function

Transforming growth factor (TGF)-beta is a potent inhibitor of adipocyte differentiation. To identify which adipocyte transcription factors might be targeted by TGF-beta, we overexpressed key adipogenic transcription factors, C/EBPbeta, C/EBPdelta, or peroxisome proliferator-activated receptor (PPAR) gamma in NIH3T3 cells and tested the ability of TGF-beta to block adipogenesis. We show that TGF-beta inhibits adipocyte differentiation driven by either C/EBPbeta or C/EBPdelta without affecting C/EBP protein expression levels, suggesting that these C/EBPs are a direct target of TGF-beta action. Because TGF-beta inhibits adipogenesis by signaling through Smad3, we examined physical and functional interactions of Smad3 and Smad4 with C/EBPbeta, C/EBPdelta, and PPARgamma2. C/EBPbeta and C/EBPdelta were found to physically interact with Smad3 and Smad4, and Smad3 cooperated with Smad4 and TGF-beta signaling to repress the transcriptional activity of C/EBPs. Thus, repression of the activity of C/EBPs by Smad3/4 at C/EBP binding sites inhibited transcription from the PPARgamma2 and leptin promoters. In contrast, PPARgamma interacted only very weakly with Smad3 and its transcriptional activity was not repressed by Smad3/4 or in response to TGF-beta. Smad3/4 did not reduce the ability of C/EBP to bind to its cognate DNA sequence, but repressed transcription by inhibiting the transactivation function of C/EBP.

The Regulation and Activation of Ciliary Neurotrophic Factor Signaling Proteins in Adipocytes

Ciliary neurotrophic factor (CNTF) is primarily known for its roles as a lesion factor released by the ruptured glial cells that prevent neuronal degeneration. However, CNTF has also been shown to cause weight loss in a variety of rodent models of obesity/type II diabetes, whereas a modified form also causes weight loss in humans. CNTF administration can correct or improve hyperinsulinemia, hyperphagia, and hyperlipidemia associated with these models of obesity. In order to investigate the effects of CNTF on fat cells, we examined the expression of CNTF receptor complex proteins (LIFR, gp130, and CNTFRalpha) during adipocyte differentiation and the effects of CNTF on STAT, Akt, and MAPK activation. We also examined the ability of CNTF to regulate the expression of adipocyte transcription factors and other adipogenic proteins. Our studies clearly demonstrate that the expression of two of the three CNTF receptor complex components, CNTFRalpha and LIFR, decreases during adipocyte differentiation. In contrast, gp130 expression is relatively unaffected by differentiation. In addition, preadipocytes are more sensitive to CNTF treatment than adipocytes, as judged by both STAT 3 and Akt activation. Despite decreased levels of CNTFRalpha expression in fully differentiated 3T3-L1 adipocytes, CNTF treatment of these cells resulted in a time-dependent activation of STAT 3. Chronic treatment of adipocytes resulted in a substantial decrease in fatty-acid synthase and a notable decline in SREBP-1 levels but had no effect on the expression of peroxisome proliferator-activated receptor gamma, acrp30, adipocyte-expressed STAT proteins, or C/EBPalpha. However, CNTF resulted in a significant increase in IRS-1 expression. CNTFRalpha receptor expression was substantially induced in the fat pads of four rodent models of obesity/type II diabetes as compared with lean littermates. Moreover, we demonstrated that CNTF can activate STAT 3 in adipose tissue and skeletal muscle in vivo. In summary, CNTF affects adipocyte gene expression, and the specific receptor for this cytokine is induced in rodent models of obesity/type II diabetes.

Adipose tissue gene expression in patients with a loss of function mutation in the leptin receptor.

In order to maintain body weight regulation, leptin directly or indirectly signals nutritional changes to key organs, but little is known about its target genes in human adipose tissue. Leptin receptor loss of function is a unique way to explore the role of leptin in the regulation of adipose tissue. We studied the consequences of the absence of leptin signaling on adipocyte gene expression in two girls with a mutation in the leptin receptor. The expression levels of the ob gene and adipocyte transcription factors (SREBP1c, C/EPBalpha, beta, PPARgamma1, gamma2) were quantified by RT-PCR in subcutaneous adipose tissue of these patients and of 10 morbidly obese women. Ob mRNA levels in subjects lacking the leptin receptor were not overexpressed but were in the range that could be expected from their BMI (58 and 26 amol/ micro g total mRNA, range in obese women: 26-69). Expression of the five transcription factors was also in the same range in the affected patients and in morbidly obese women (7.7 and 6.8 amol/ micro g total mRNA, range: 2.2-9.4 for SREBP1c, 159 and 51 range: 51-406 for C/EPBalpha, 6.1 and 3.3 range: 2.4-24.8 for C/EPBbeta, 16.7 and 27.4 range: 9.4-29.7 for PPARgamma1 and 1.7 and 5.4 amol/ micro g total mRNA range: 1.7-8.8 for PPARgamma2). Significant correlation was found between the mRNA levels of leptin and PPARgamma2 and leptin and C/EBPalpha whereas no correlation was observed between leptin and SREBP1c, PPARgamma1, or C/EBPbeta mRNA levels. In patients lacking leptin signaling, the fact that ob gene expression is adequately adapted to their body fat mass argues against a direct negative feedback loop in the regulation of leptin expression in humans. The normal expression of several transcription factors, known to be dependent of the nutritional status, suggests that leptin is not a major contributor of their in vivo transcriptional regulation in human adipose tissue.