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It is generally assumed that white adipocytes arise from resident adipose tissue mesenchymal progenitor cells. We challenge this paradigm by defining a hematopoietic origin for both the de novo development of a subset of white adipocytes in adults and a previously uncharacterized adipose tissue resident mesenchymal progenitor population. Lineage and cytogenetic analysis revealed that bone marrow progenitor (BMP)-derived adipocytes and adipocyte progenitors arise from hematopoietic cells via the myeloid lineage in the absence of cell fusion. Global gene expression analysis indicated that the BMP-derived fat cells are bona fide adipocytes but differ from conventional white or brown adipocytes in decreased expression of genes involved in mitochondrial biogenesis and lipid oxidation, and increased inflammatory gene expression. The BMP-derived adipocytes accumulate with age, occur in higher numbers in visceral than in subcutaneous fat, and in female versus male mice. BMP-derived adipocytes may, therefore, account in part for adipose depot heterogeneity and detrimental changes in adipose metabolism and inflammation with aging and adiposity. bone marrow | hematopoietic | stem cell doi/ 10.1073/pnas.1003512107

Fibroblast growth factor 21 (FGF21) has been identified as a potent metabolic regulator. Administration of recombinant FGF21 protein to rodents and rhesus monkeys with diet-induced or genetic obesity and diabetes exerts strong antihyperglycemic and triglyceride-lowering effects and reduction of body weight. Despite the importance of FGF21 in the regulation of glucose, lipid, and energy homeostasis, the mechanisms by which FGF21 functions as a metabolic regulator remain largely unknown. Here we demonstrate that FGF21 regulates energy homeostasis in adipocytes through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), resulting in enhanced mitochondrial oxidative function. AMPK phosphorylation levels were increased by FGF21 treatment in adipocytes as well as in white adipose tissue from ob/ob mice. FGF21 treatment increased cellular [NAD.sup.+] levels, leading to activation of SIRT1 and deacetylation of its downstream targets, peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] (PGC-1[alpha]) and histone 3. Activation of AMPK and SIRT1 by FGF21 in adipocytes enhanced mitochondrial oxidative capacity as demonstrated by increases in oxygen consumption, citrate synthase activity, and induction of key metabolic genes. The effects of FGF21 on mitochondrial function require serine/ threonine kinase 11 (STK11/LKB1), which activates AMPK. Inhibition of AMPK, SIRT1, and PGC-1[alpha] activities attenuated the effects of FGF21 on oxygen consumption and gene expression, indicating that FGF21 regulates mitochondrial activity and enhances oxidative capacity through an AMPK--SIRT1-PGC1[alpha]-dependent mechanism in adipocytes. adipocytes | diabetes | obesity | mitochondrial function doi/10.1073/pnas.1006962107

Adipose tissue inflammation is linked to the pathogenesis of insulin resistance. In addition to exerting death-promoting effects, the death receptor Fas (also known as CD95) can activate inflammatory pathways in several cell lines and tissues, although little is known about the metabolic consequence of Fas activation in adipose tissue. We therefore sought to investigate the contribution of Fas in adipocytes to obesity-associated metabolic dysregulation. Fas expression was markedly increased in the adipocytes of common genetic and diet-induced mouse models of obesity and insulin resistance, as well as in the adipose tissue of obese and type 2 diabetic patients. Mice with Fas deficiency either in all cells or specifically in adipocytes (the latter are referred to herein as AFasKO mice) were protected from deterioration of glucose homeostasis induced by high-fat diet (HFD). Adipocytes in AFasKO mice were more insulin sensitive than those in wild-type mice, and mRNA levels of proinflammatory factors were reduced in white adipose tissue. Moreover, AFasKO mice were protected against hepatic steatosis and were more insulin sensitive, both at the whole-body level and in the liver. Thus, Fas in adipocytes contributes to adipose tissue inflammation, hepatic steatosis, and insulin resistance induced by obesity and may constitute a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes., Introduction White adipose tissue (WAT) has been recognized as an important endocrine organ secreting different hormone-like factors (adipokines), FFAs, and cytokines, thereby regulating metabolism locally and systemically (1). In obesity, [...]

Increases in key components of adipogenesis and lipolysis pathways correlate at the population-averaged level during adipogenesis. However, differentiating preadipocytes are highly heterogeneous in cellular and lipid droplet (LD) morphologies, and the degree to which individual cells follow population-averaged trends is unclear. In this study, we analyze the molecular heterogeneity of differentiating 3T3-L1 preadipocytes using immuno-fluorescence microscopy. Unexpectedly, we only observe a small percentage of cells with high simultaneous expression of markers for adipogenesis (peroxisome proliferator-activated receptor [gamma] [PPAR[gamma]], CCAAT/enhancer-binding protein [alpha], and adiponectin) and lipid accumulation (hormone-sensitive lipase, perilipin A, and LDs). Instead, we identify subpopulations of cells with negatively correlated expressions of these readouts. Acute perturbation of adipocyte differentiation with PPAR[gamma], agonists, forskolin, and fatty acids induced subpopulation-specific effects, including redistribution of the percentage of cells in observed subpopulations and differential expression levels of PPAR[gamma]. Collectively, our results suggested that heterogeneity observed during 3T3-L1 adipogenesis reflects a dynamic mixture of subpopulations with distinct physiological states. doi/10.1083/jcb.200904140

The adipocyte-derived hormone leptin maintains energy balance by acting on hypothalamic leptin receptors (Leprs) that act on the signal transducer and activator of transcription 3 (Stat3) (1-4). Although disruption of Lepr-Stat3 signaling promotes obesity in mice, other features of Lepr function, such as fertility, seem normal, pointing to the involvement of additional regulators. Here we show that the cyclic AMP responsive element-binding protein-1 (Creb1)-regulated transcription coactivator-1 (Crtc1) is required for energy balance and reproduction-[Crtc1.sup.-/-] mice are hyperphagic, obese and infertile. Hypothalamic Crtc1 was phosphorylated and inactive in leptin-deficient oblob mice, while leptin administration increased amounts of dephosphorylated nuclear Crtc1. Dephosphorylated Crtc1 stimulated expression of the Cartptand Kiss1 genes, which encode hypothalamic neuropeptides that mediate leptin's effects on satiety and fertility (5-7). Crtc1 overexpression in hypothalamic cells increased Cartptand Kiss1 gene expression, whereas Crtc1 depletion decreased it. Indeed, leptin enhanced Crtc1 activity over the Cartpt and Kiss1 promoters in cells overexpressing Lepr, and these effects were disrupted by expression of a dominant-negative Creb1 polypeptide. As leptin administration increased recruitment of hypothalamic Crtc1 to Cartpt and Kiss1 promoters, our results indicate that the Creb1-Crtc1 pathway mediates the central effects of hormones and nutrients on energy balance and fertility., Crtcs (also known as transducers of regulated CREB activity, or TORCs) are latent cytoplasmic coactivators that shuttle to the nucleus in response to cyclic AMP (cAMP) and calcium signals (8, [...]

White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPAR[gamma] in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity., Introduction Obesity is associated with various complications such as insulin resistance, type 2 diabetes, dyslipidemia, and atherosclerosis (1), (2). It results from an imbalance between energy intake and energy expenditure [...]

Storage of energy as triglyceride in large adipose-specific lipid droplets is a fundamental need in all mammals. Efficient sequestration of fat in adipocytes also prevents fatty acid overload in skeletal muscle and liver, which can impair insulin signaling. Here we report that the Cide domain-containing protein Cidea, previously thought to be a mitochondrial protein, colocalizes around lipid droplets with perilipin, a regulator of lipolysis. Cidea-GFP greatly enhances lipid droplet size when ectopically expressed in preadipocytes or COS cells. These results explain previous findings showing that depletion of Cidea with RNAi markedly elevates lipolysis in human adipocytes. Like perilipin, Cidea and the related lipid droplet protein Cidec/FSP27 are controlled by peroxisome proliferator-activated receptor [gamma] (PPAR[gamma]). Treatment of lean or obese mice with the PPAR[gamma] agonist rosiglitazone markedly up-regulates Cidea expression in white adipose tissue (WAT), increasing lipid deposition. Strikingly, in both omental and s.c. WAT from BMI-matched obese humans, expression of Cidea, Cidec/FSP27, and perilipin correlates positively with insulin sensitivity (HOMA-IR index). Thus, Cidea and other lipid droplet proteins define a novel, highly regulated pathway of triglyceride deposition in human WAT. The data support a model whereby failure of this pathway results in ectopic lipid accumulation, insulin resistance, and its associated comorbidities in humans. adipocyte | Cide | diabetes | fat droplet | fat metabolism

In the development of obesity, the source of excess energy may influence appetite and metabolism. To determine the effects of differences in diet composition in obesity, mice were fed either a high-carbohydrate diet (HC; 10% fat energy) or a high-fat energy-restricted diet (HFR; 60% fat energy) over 18 wk in weight-matched groups of mice. To identify obesity-associated genes with persistently altered expression following weight reduction, mice were fed either a standard low-fat diet (LF; 10% fat energy), an unrestricted high-fat diet (HF; 60% fat energy), or a HF diet followed by weight reduction (WR). Mice fed a HF diet had significantly greater gonadal fat mass and higher whole blood glucose concentrations than mice fed an HC diet. Of the mice fed a high-fat diet, total body weight and serum insulin concentrations were greater in HF than in HFR. Microarray analysis revealed that HF vs. HC feeding resulted in global differences in adipocyte gene expression patterns. Although we identified genes whose expression was altered in both moderately and severely obese mice, there were also a large number of genes with altered expression only in severe obesity. Formerly obese, WR mice did not differ significantly from lean controls in total body weight or physiological measures. However, microarray analysis revealed distinctly different patterns of adipocyte gene expression. Furthermore, there were 398 genes with altered expression in HF mice that persisted in WR mice. Genes with persistently altered expression following obesity may play a role in rebound weight gain following weight reduction.

Recently, we characterized tumor suppressor candidate 5 (Tusc5) as an adipocyte-neuron PPAR[gamma] target gene. Our objective herein was to identify additional genes that display distinctly high expression in fat and neurons, because such a pattern could signal previously uncharacterized functional pathways shared in these disparate tissues. [gamma]-Synuclein, a marker of peripheral and select central nervous system neurons, was strongly expressed in white adipose tissue (WAT) and peripheral nervous system ganglia using bioinformatics and quantitative PCR approaches. [gamma]-Synuclein expression was determined during adipogenesis and in subcutaneous (SC) and visceral adipose tissue (VAT) from obese and nonobese humans. [gamma]-Synuclein mRNA increased from trace levels in preadipocytes to high levels in mature 3T3-L1 adipocytes and decreased -50% following treatment with the PPAR[gamma] agonist GW1929 (P < 0.01). Because [gamma]-synuclein limits growth arrest and is implicated in cancer progression in nonadipocytes, we suspected that expression would be increased in situations where WAT plasticity/adipocyte turnover are engaged. Consistent with this postulate, human WAT 7-synuclein mRNA levels consistently increased in obesity and were higher in SC than in VAT; i.e. they increased ~1-7-fold in obese Pima Indian adipocytes (P = 0.003) and ~2-fold in SC and VAT of other obese cohorts relative to nonobese subjects. Expression correlated with leptin transcript levels in human SC and VAT (r = 0.887; P < 0.0001 ; n = 44). [gamma]-Synuclein protein was observed in rodent and human WAT but not in negative control liver. These results are consistent with the hypothesis that [gamma]-synuclein plays an important role in adipocyte physiology.

We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) causes human adipocyte delipidation, insulin resistance, and inflammation in part by attenuating PPAR[gamma] target gene expression. We hypothesized that CLA antagonizes the activity of PPAR[gamma] in an isomer-specific manner. 10,12 CLA, but not cis-9, trans-11 (9,11 ) CLA, suppressed ligand-stimulated activation of a peroxisome proliferator response element-luciferase reporter. This decreased activation of PPAR[gamma] by 10,12 CLA was accompanied by an increase in PPAR[gamma] and extracellular signal-related kinase (ERK) 1/2 phosphorylation, followed by decreased PPAR[gamma], protein levels. To investigate if 10,12 CLA-mediated delipidation was preventable with a PPAR[gamma] ligand (BRL), cultures were treated for 1 wk with 10,12 CLA or 10,12 CLA + BRL and adipogenic gene and protein expression, glucose uptake, and triglyceride (TG) were measured. BRL cosupplementation completely prevented 10,12 CLA suppression of adipocyte fatty acid-binding protein, lipoprotein lipase, and perilipin mRNA levels without preventing reductions in PPAR[gamma] or insulin-dependent glucose transporter 4 (GLUT4) expression, glucose uptake, or TG. Lastly, we investigated the impact of CLA withdrawal in the absence or presence of BRL for 2 wk. CLA withdrawal did not rescue CLA-mediated reductions in adipogenic gene and protein expression. In contrast, BRL supplementation for 2 wk following CLA withdrawal rescued mRNA levels of PPAR[gamma] target genes. However, the levels of PPAR[gamma] and GLUT4 protein and TG were only partially rescued by BRL. Collectively, we demonstrate for the first time, to our knowledge, that 10,12 CLA antagonizes ligand-dependent PPAR[gamma] activity, possibly via PPAR[gamma] phosphorylation by ERK.

The functional trafficking steps used by soluble NSF attachment protein receptor (SNARE) proteins have been difficult to establish because of substantial overlap in subcellular localization and because in vitro SNARE-dependent binding and fusion reactions can be promiscuous. Therefore, to functionally identify the site of action of the vesicle-associated membrane protein (VAMP) family of R-SNAREs, we have taken advantage of the temporal requirements of adipocyte biosynthetic sorting of a dual-tagged GLUT4 reporter (myc-GLUT4-GFP) coupled with small interfering RNA gene silencing. Using this approach, we confirm the requirement of VAMP2 and VAMP7 for insulin and osmotic shock trafficking from the vesicle storage sites, respectively, and fusion with the plasma membrane. Moreover, we identify a requirement for VAMP4 for the initial biosynthetic entry of GLUT4 from the Golgi apparatus into the insulin-responsive vesicle compartment, VAMP8, for plasma membrane endocytosis and VAMP2 for sorting to the specialized insulin-responsive compartment after plasma membrane endocytosis.

The ability to store fat in the form of cytoplasmic triglyceride droplets is conserved from Saccharomyces cerevisiae to humans. Although much is known regarding the composition and catabolism of lipid droplets, the molecular components necessary for the biogenesis of lipid droplets have remained obscure. Here we report the characterization of a conserved gene family important for lipid droplet formation named fat-inducing transcript (FIT). FIT1 and FIT2 are endoplasmic reticulum resident membrane proteins that induce lipid droplet accumulation in cell culture and when expressed in mouse liver, shRNA silencing of FIT2 in 3T3-LI adipocytes prevents accumulation of lipid droplets, and depletion of FIT2 in zebrafish blocks diet-induced accumulation of lipid droplets in the intestine and liver, highlighting an important role for FIT2 in lipid droplet formation in vivo. Together these studies identify and characterize a conserved gene family that is important in the fundamental process of storing fat. adipocytes | diabetes | FIT | obesity | triglyceride

The E2F and pocket protein families are known to play an important role in the regulation of both cellular proliferation and terminal differentiation. In this study, we have used compound E2F and pocket protein mutant mouse embryonic fibroblasts to dissect the role of these proteins in adipogenesis. This analysis shows that loss of E2F4 allows cells to undergo spontaneous differentiation. The ability of E2F4 to prevent adipogenesis seems to be quite distinct from the known properties of E2F. First, it can be separated from any change in either E2F-responsive gene expression or cell cycle regulation. Second, it is a specific property of E2F4, and not other E2Fs, and it occurs independently of E2F4's ability to interact with pocket proteins. In addition, E2F4 loss does not override the differentiation defect resulting from pRB loss even though it completely suppresses the proliferation defect of [Rb.sup.-/-] mouse embryonic fibroblasts. This finding definitively separates the known, positive role of pRB in adipogenesis from its cell cycle function and shows that this pocket protein is required to act downstream of E2F4 in the differentiation process.

Mesenchymal cells can differentiate into osteoblasts, adipocytes, myoblasts, or chondroblasts. Whether mesenchymal cells that have initiated differentiation along one lineage can transdifferentiate into another is largely unknown. Using 3T3-F442A preadipocytes, we explored whether extracellular signals could redirect their differentiation from adipocyte into osteoblast. 3T3-F442A cells expressed receptors and Smads required for bone morphogenetic protein (BMP) signaling. BMP-2 increased proliferation and induced the early osteoblast differentiation marker alkaline phosphatase, yet only mildly affected adipogenic differentiation. Retinoic acid inhibited adipose conversion and cooperated with BMP-2 to enhance proliferation, inhibit adipogenesis, and promote early osteoblastic differentiation. Expression of BMP-RII together with BMP-RIA or BMP-RIB suppressed adipogenesis of 3T3-F442A cells and promoted full osteoblastic differentiation in response to retinoic acid. Osteoblastic differentiation was characterized by induction of cbfa1, osteocalcin, and collagen I expression, and extracellular matrix calcification. These results indicate that 3T3-F442A preadipocytes can be converted into fully differentiated osteoblasts in response to extracellular signaling cues. Furthermore, BMP and retinoic acid signaling cooperate to stimulate cell proliferation, repress adipogenesis, and promote osteoblast differentiation. Finally, BMP-RIA and BMP-RIB induced osteoblast differentiation and repressed adipocytic differentiation to a similar extent.

Results demonstrate that between the two splice variants of the peroxisome proliferator activated receptor gamma, gamma1 and gamma2, the latter is critical for adipogenesis. Data reveal that in 3T3-L1 cells, expressing engineered zinc finger repressor proteins, a reduction in the expression of gamma2 reduces adipogenesis, whereas gamma1 reduction shows no effect on adipogenesis.

This article discusses the crucial role of the gamma2 isoform of the peroxisome proliferator activated receptor gamma, a member of the nuclear hormone receptors family, in regulating adipogenesis. A rational intervention calls for understanding the adipocyte genes, development, and function with gamma2 receptor serving as a target in adipogenesis.

Research has been conducted on the chromatin remodeling important for the promoter activation during cellular lineage commitment and differentiation. The ability of the C/EBP(alpha) transcription factor to direct adipocyte uncommitted fibroblast precursor differentiation and to activate the SWI/SNF-dependent myeloid-specific genes has been investigated and the results are reported.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), an essential regulator of adipogenesis and a modulator of fat cell function, is discussed in this review article. Topics include adipogenic cell lines, gain-of-function and loss-of-function experiments, CCAAT/enhancer binding proteins (C/EBPs), and roles in fully differentiated mature adipocytes. TZD-mediated stimulation of PPAR-gamma helps insulin resistance. It appears that normal amounts of PPAR-gamma activity, under some circumstances, promote disease. Both its agonists and its antagonists might be useful clinically. Areas of interest for the future include the role of dietary fats as precursors to PPAR-gamma ligands.

Mitotic clonal expansion of adipocyte differentiation is discussed with consideration of activation and centromeric localization of CCAAT/enhancer-binding proteins. These occur in the expansion. It has been shown that C/EBP-beta and C/EBP-delta, transcription factors, cannot bind to the C/EBP regulatory element in the C/EBP-alpha promoter. As the preadipocytes enter S phase and the mitotic clonal expansion begins, C/EBP-beta/delta start to acquire capacity to bind the C/EBP regulatory element and become centromere-associated.

Two strains of obese mice have been shown to have defects in the gene encoding the protein leptin, which is secreted by adipocytes. An examination of two severely obese children shows that they are both homozygous for a single-nucleotide deletion in the gene encoding leptin. Their obesity provides the first evidence that leptin is important in regulating the energy balance of humans.

Lack of leptin (ob) protein causes obesity in mice. The leptin gene product is important for normal regulation of appetite and metabolic rate and is produced exclusively by adipocytes. Leptin mRNA was induced during the adipose conversion of 3T3-L1 cells, which are useful for studying adipocyte differentiation and function under controlled conditions. We studied leptin regulation by antidiabetic thiazolidinedione compounds, which are ligands for the adipocyte-specific nuclear receptor peroxisome proliferator-activated receptor [Gamma] ([PPAR.sub.[Gamma]]) that regulates the transcription of other adipocyte-specific genes. Remarkably, leptin gene expression was dramatically repressed within a few hours after thiazolidinedione treatment. The [ED.sub.50] for inhibition of leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its [K.sub.d] for binding to [PPAR.sub.[Gamma]]. The relatively weak, nonthiazolidinedione PPAR activator WY 14,643 also inhibited leptin expression, but was [approximately equal to]1000 times less potent than BRL49653. These results indicate that antidiabetic thiazolidinediones down-regulate leptin gene expression with potencies that correlate with their abilities to bind and activate [PPAR.sub.[Gamma]].

SV40 early region genes were placed under the control of the regulatory region from the aP2 gene and created transgenic mice to create a model system for studying adipocyte transformation. Brown fat tumors or hibernomas developed in transgenic mice as early as one day after birth which expressed brown fat-specific uncoupling protein gene as well as the aP2 gene. These results suggest that regulation of the UCP thermogenic gene can be studied in an established cell line.

Research has been conducted on adipocytes and myocytes derived from mesenchymal precursor. The authors report that Rho GTPase is a modylator of IGF-1 signals which affect adipogenessis and myogenesis cell fate decision.

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

Retinoid X receptor [Alpha] (RXR[Alpha]) is involved in multiple signaling pathways, as a heterodimeric partner of several nuclear receptors. To investigate its function in energy homeostasis, we have selectively ablated the RXR[Alpha] gene in adipocytes of 4-week-old transgenic mice by using the tamoxifen-inducible Cre-[ER.sup.T2] recombination system. Mice lacking RXR[Alpha] in adipocytes were resistant to dietary and chemically induced obesity and impaired in fasting-induced lipolysis. Our results also indicate that RXR[Alpha] is involved in adipocyte differentiation. Thus, our data demonstrate the feasibility of adipocyte-selective temporally controlled gene engineering and reveal a central role of RXR[Alpha] in adipogenesis, probably as a heterodimeric partner for peroxisome proliferator-activated receptor [Gamma]. peroxisome proliferator-activated receptor [Gamma] | conditional mutagenesis | obesity | fasting | adipocyte fatty acid binding protein

CCAAT enhancer-binding protein-[Alpha] (C/EBP[Alpha]) functions as a pleiotropic transcriptional activator of adipocyte genes during adipogenesis. Nuclear factor C/EBP undifferentiated protein (CUP), an isoform of activator protein-2[Alpha] (AP-2[Alpha]), binds to repressive elements in the C/EBP[Alpha] gene promoter, silencing the gene until late in the differentiation program. The CUP regulatory element overlaps a Sp (GT-box) element in the promoter to which Sp3 (or Sp1) can bind. Binding by Sp3 or Sp1 and CUP/AP2-[Alpha] is mutually exclusive. Sp3 is a strong transcriptional activator of the C/EBP[Alpha] gene promoter in 3T3-L1 preadipocytes and Schneider cells, this activation being repressed by CUP/AP-2[Alpha]. Sp3 is expressed throughout differentiation, whereas CUP/AP-2[Alpha], which is expressed only by preadipocytes, is down-regulated during differentiation coincident with transcription of the C/EBP[Alpha] gene. Thus, CUP/AP-2[Alpha] delays access of Sp3 to the Sp regulatory element, preventing premature expression of C/EBP[Alpha] and thereby interference by C/EBP[Alpha] (which is antimitotic) with mitotic clonal expansion, an essential early event in the differentiation program. 3T3-L1 adipocyte | CEBP[Alpha] undifferentiated protein | AP-2 | Sp1 | Sp3

The human homologue of the murine obesity gene, agouti, is expressed in adipose tissue. We have shown that recombinant agouti protein regulates adipocyte lipogenesis and lipolysis coordinately and promotes lipid storage via a [Ca.sup.2+]-dependent mechanism in vitro, which may contribute to agouti-induced obesity. However, little is known about agouti's physiologic function in humans. We first studied the agouti content in human mature adipocytes vs. preadipocytes. The agouti content of human mature adipocytes was five times as abundant as in preadipocytes (19.18 [+ or -] 2.46 vs. 4.07 [+ or -] 0.51 pg/[micro]g protein, P [is less than] 0.005), suggesting that agouti is up-regulated during adipocyte differentiation. We next studied the relationship of agouti mRNA and protein to fatty acid synthase (FAS) mRNA and activity in adipose tissue obtained from nonobese and mildly obese patients (body mass index range, 21-31 kg/[m.sup.2]). Agouti protein was correlated with FAS activity (r = 0.782, P [is less than] 0.005). Similarly, human adipose tissue agouti mRNA level was also correlated with FAS mRNA level (r = 0.846, P [is less than] 0.001). These data suggest that agouti may be another adipocyte-produced factor that modulates adipocyte lipid metabolism via a paracrine/autocrine mechanism. J. Nutr. 130: 2478-2481, 2000. KEY WORDS: * agouti * fatty acid synthase * adipose tissue * adipocyte differentiation * obesity * humans

Conjugated linoleic acids (CLA) are a group of positional and geometric conjugated dienoic isomers of linoleic acid. The objective of this study was to determine the effects of the cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on lipid composition and gene expression during the differentiation of mouse 3T3-L1 preadipocytes. Treatment of differentiating 3T3-L1 preadipocytes with trans-10,cis-12 conjugated linoleic acid (CLA) resulted in a dose-dependent decrease in the expression of the stearoyl-CoA desaturase 1 gene (SCD1). The expression of other adipocyte genes such as adipose P2 (aP2), fatty acid synthase (FAS), SCD2 and the key adipogenic transcription factors, peroxisome proliferator-activated receptor [Gamma]2 (PPAR[Gamma]2) and CCAAT enhancer binding protein [Alpha] (C/EBP[Alpha]), remained elevated. Cells treated with trans-10,cis-12 CLA exhibited smaller lipid droplets, with reduced levels of the major monounsaturated fatty acids, palmitoleate and oleate. By contrast, the cis-9,trans-11 isomer did not alter adipocyte gene expression. Repression of the stearoyl-CoA desaturase gene expression in adipocytes by the trans-10,cis-12 isomer may contribute to the mechanisms by which CLA reduces body fat in mice. J. Nutr. 130: 1920-1924, 2000. KEY WORDS: * mouse adipocytes * differentiation * conjugated linoleic acid.

Antidiabetic and hypoglycemic drugs were recently reported to enhance adipose differentiation of 3T3-L1 preadipocytes. In this experiment, the effect of soybean constituents on adipose differentiation was investigated by using mouse 3T3-L1 preadipocytes. Active constituents were purified and identified. 3T3-L1 cells were grown as monolayer cultures at 37 [degrees] C in DME-medium supplemented by 10% fetal bovine serum under an atmosphere of 5% [CO.sub.2]/95% air. Confluent cell cultures were then treated and converted to adipocytes by culture in the presence of insulin and soybean extract samples. After ~10 d, glycerol-3-phosphate dehydrogenase activity and triglyceride accumulation were measured as markers of adipose differentiation. Insulin-sensitive glucose uptake and lipoprotein lipase activity were also assayed in the treated cells. Crude soybean extract was found to enhance the differentiation. Active constituents were purified and identified as isoflavones (e.g., daidzein or genistein). Aglycones were more active than glycosides. In the daidzein-treated cells, insulin-sensitive glucose uptake and lipoprotein lipase activity were increased. In this experiment, it is proposed that the activation of adipose function by newly differentiated adipocytes seems to be an important factor in explaining the mechanism for the effect of soybean in the prevention and treatment of chronic diseases.