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Endogenous adipocyte apolipoprotein E (apoE) plays an important role in adipocyte lipoprotein metabolism and lipid flux. A potential role for hyperglycemia in regulating adipocyte apoE expression and triglyceride metabolism was examined. Exposure of adipocytes to high glucose or advanced glycosylation end product-BSA significantly suppressed apoE mRNA and protein levels. This suppression was significantly attenuated by antioxidants or inhibitors of the NF-KB transcription pathway. Hyperglycemia in vivo led to adipose tissue oxidant stress and significant reduction in adipose tissue and adipocyte apoE mRNA level. Incubation with antioxidant in organ culture completely reversed this suppression. Hyperglycemia also reduced adipocyte triglyceride synthesis, and this could be completely reversed by adenoviral-mediated increases in apoE. To more specifically evaluate an in vivo role for adipocyte apoE expression on organismal triglyceride distribution in vivo, WT or apoE knockout (EKO) adipose tissue was transplanted in EKO recipient mice. After 12 wk, WT adipocytes transplanted in EKO mice accumulated more triglyceride compared with transplanted EKO adipocytes. In addition, EKO recipients of WT adipose tissue had reduced hepatic triglyceride content compared with EKO recipients transplanted with EKO adipose tissue. Our results demonstrate that hyperglycemia and advanced glycosylation end products suppress the expression of adipocyte apoE in vitro and in vivo and thereby reduce adipocyte triglyceride synthesis. In vivo results using adipose tissue transplantation suggest that reduction of adipocyte apoE, and subsequent reduction of adipocyte triglyceride accumulation, could influence lipid accumulation in nonadipose tissue. adipocytes; adipose tissue; adipose tissue transplantation; hyperglycemia; apolipoprotein E; oxidant stress doi: 10.1152/ajpendo.00273.2010.

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

In this study, we explored the roles of microRNAs in adipocyte differentiation and metabolism. We first knocked down Argonaute2 (Ago2), a key enzyme in the processing of micro-RNAs (miRNAs), to investigate a potential role for miRNAs in adipocyte differentiation and/or metabolism. Although we did not observe dramatic differences in adipogenesis between Ago2 knock-down and control 3T3-L1 cells, incorporation of [[sup.14]C]glucose or acetate into triacylglycerol, and steady-state levels of triacyglycerol were all reduced, suggesting a role for miRNAs in adipocyte metabolism. To study roles of specific miRNAs in adipocyte biology, we screened for miRNAs that are differentially expressed between preadipocytes and adipocytes for the 3T3-L1 and ST2 cell lines. Distinct subsets of miRNAs decline or increase during adipocyte conversion, whereas most miRNAs are not regulated. One locus encoding two miRNAs, 378/378', contained within the intron of PGC-1[beta] is highly induced during adipogenesis. When overexpressed in ST2 mesenchymal precursor cells, [miRNA378/378.sup.*] increases the size of lipid droplets and incorporation of [[sup.14]C]acetate into triacyl-glycerol. Although protein and mRNA expression levels of C/EBP[alpha], C/EBP[beta], C/EBP[delta], and PPAR[gamma]1 are unchanged, microarray and quantitative RT-PCR analyses indicate that a set of lipogenic genes are upregulated, perhaps due to increased expression of PPAR[gamma]2. Knockdown of miRNA378 and/or [miRNA378.sup.*] decreases accumulation of triacylglycerol. Interestingly, we made the unexpected finding that [miRNA378/378.sup.*] specifically increases transcriptional activity of C/EBP[alpha] and C/EBP[beta] on adipocyte gene promoters. adipogenesis; PPAR[gamma], coactivator-l[beta]; micro-RNA doi: 10.1152/ajpendo.00179.2010.

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

Fat distribution changes with aging. Inherent changes in fat cell progenitors may contribute because fat cells turn over throughout life. To define mechanisms, gene expression was profiled in preadipocytes cultured from epididymal and perirenal depots of young and old rats. 8.4% of probe sets differed significantly between depots, particularly developmental genes. Only 0.02% differed with aging, despite using less stringent criteria than for comparing depots. Twenty-five genes selected based on fold change with aging were analyzed in preadipocytes from additional young, middle-aged, and old animals by polymerase chain reaction. Thirteen changed significantly with aging, 13 among depots, and 9 with both. Genes involved in inflammation, stress, and differentiation changed with aging, as occurs in fat tissue. Age-related changes were greater in perirenal than epididymal preadipocytes, consistent with larger declines in replication and adipogenesis in perirenal preadipocytes. Thus, age-related changes in preadipocyte gene expression differ among depots, potentially contributing to fat redistribution and dysfunction. Key Words: Aging--Preadipocyte--Fat cell progenitor. doi: 10.1093/gerona/glp213

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

Salt-inducible kinase 2 (SIK2) is expressed abundantly in adipose tissues and represses cAMP-response element-binding protein (CREB)-mediated gene expression by phosphorylating the coactivator transducer of regulated CREB activity (TORC2). Phosphorylation at [Ser.sup.587] of SIK2 diminishes its TORC2 phosphorylation activity. In 3T3-LI white adipocytes, SIK2 downregulates lipogenic gene in response to nutritional stresses. To investigate the impact of SIK2 on the function of brown adipose tissue (BAT), we used T37i brown adipocytes, mice with diet-induced obesity, and SIK2 mutant (S587A) transgenic mice. When T37i adipocytes were treated with insulin, the levels of peroxisome proliferator-activated receptor-coactivator-l[alpha] (PGC-1[alpha]) and uncoupling protein-1 (UCP-1) mRNA were increased, and the induction was inhibited by overexpression of SIK2 (S587A) mutant or dominant-negative CREB. Insulin enhanced SIK2 phosphorylation at [Ser.sup.587], which was accompanied by decrease in phospho-TORC2. Similarly, the decrease in the level of SIK2 phosphorylation at [Ser.sup.587] was observed in the BAT of mice with diet-induced obesity, which was negatively correlated with TORC2 phosphorylation. To confirm the negative correlation between SIK2 phosphorylation at [Ser.sup.587] and TORC2 phosphorylation in BAT, SIK2 mutant (S587A) was overexpressed in adipose tissues by using the adipocyte fatty acid-binding protein 2 promoter. The expression of recombinant SIK2 (S587A) was restricted to BAT, and the levels of phospho-TORC2 were elevated in BAT of transgenic mice. Male transgenic mice developed high-fat diet-induced obesity, and their BAT expressed low levels of PGC-1[alpha] and UCP-1 mRNA, suggesting that SIK2-TORC2 cascade may be important for the regulation of PGC-1[alpha] and UCP-1 gene expression in insulin signaling in BAT. salt-inducible kinase 2; peroxisome proliferator-activated receptorcoactivator-l[alpha]; uncoupling protein-l; brown adipocyte; adenosine 5',3'-cyclic monophosphate-response element-binding protein

Recent studies have established that vitamin D plays multiple biological roles beyond calcium metabolism; however, whether vitamin D is involved in energy metabolism is unknown. To address this question, we characterized the metabolic phenotypes of vitamin D receptor (VDR)-null mutant mice. Under a normocalcemic condition, VDR-null mice displayed less body fat mass and lower plasma triglyceride and cholesterol levels compared with wild-type (WT) mice; when placed on a high-fat diet, VDR-null mice showed a slower growth rate and accumulated less fat mass globally than WT mice, even though their food intake and intestinal lipid transport capacity were the same as WT mice. Consistent with the lower adipose mass, plasma leptin levels were lower and white adipocytes were histologically smaller in VDR-null mice than WT mice. The rate of fatty acid [beta]-oxidation in the white adipose tissue was higher, and the expression of uncoupling protein (UCP) 1, UCP2 and UCP3 was markedly upregulated in VDR-null mice, suggesting a higher energy expenditure in the mutant mice. Experiments using primary brown fat culture confirmed that 1,25-dihydroxyvitamin [D.sub.3] directly suppressed the expression of the UCPs. Consistently, the energy expenditure, oxygen consumption, and C[O.sub.2] production in VDR-null mice were markedly higher than in WT mice. These data indicate that vitamin D is involved in energy metabolism and adipocyte biology in vivo in part through regulation of [beta]-oxidation and UCP expression. adipocytes; uncoupling proteins; energy metabolism

Obesity is associated with macrophage accumulation and inflammation in adipose tissue. Macrophage-secreted factors have been reported to inhibit the differentiation of preadipocytes into adipocytes and to modulate adipogenic extracellular matrix gene expression. To enlarge our understanding of macrophages and the scope of their interactions with preadipocytes, we investigated their effect on preadipocyte survival. Acute exposure of 3T3-L1 preadipocytes to J774A.1 macrophage-conditioned medium (MacCM) stimulated platelet-derived growth factor receptor (PDGFR) tyrosine phosphorylation by 4.1-fold. There were significant increases in the phosphocontent of downstream PDGFR targets Akt and ERK1/2 (5.3-fold and 2.4-fold, respectively) that were inhibited by PDGF immunoneutralization or by the selective PDGFR inhibitor imatinib. Serum-free J774A.1-MacCM or RAW264.7-MacCM completely prevented 3T3-L1 preadipocyte apoptosis normally induced by serum deprivation. Addition of PDGF alone to serum-free control medium was sufficient to prevent 3T3-L1 preadipocyte apoptosis. Inhibition of PDGFR activation by MacCM, either by addition of imatinib or by PDGF immunodepletion of MacCM, effectively disrupted the prosurvival effect. In summary, our data indicate that MacCM promotes preadipocyte survival in a PDGF-dependent manner. platelet-derived growth factor; obesity; macrophage

The nuclear transcription factor peroxisome proliferator-activated receptor [gamma] (PPAR[gamma]) triggers adipocyte differentiation by regulating lipogenic genes. A ligand for PPAR[gamma] is necessary to activate PPAR[gamma] function. Fatty acids are potential ligands for PPAR[gamma] activation. The current experiment was designed to determine the potential for individual fatty acids to activate porcine PPAR[gamma] ectopically expressed in myoblasts. The expression of adipocyte fatty acid binding protein (aP2) and adiponectin in myoblasts stably expressing porcine PPAR[gamma] was increased when docosahexaenoic acid (DHA) was added to the adipogenic medium. The response was positively related to DHA concentration and suggests that DHA may bind to and activate porcine PPAR[gamma], leading to increased expression of aP2 and adiponectin. The conditioned media collected from myoblasts expressing PPAR[gamma] between d 3 and 6 or between d 6 and 9, but not DHA itself, activated the aP2 gene promoter-driven luciferase activity. These results suggest that a metabolite of DHA is the ligand binding to and activating porcine PPAR[gamma]. The metabolite and pathway for its production are currently unknown. Key words: adipocyte differentiation, adipocyte fatty acid-binding protein, adiponectin, docosahexaenoic acid, peroxisome proliferator-activated receptor [gamma], pig

Adipose tissue development is a process that comprises not only hypertrophy, but also hyperplasia, of adipocytes. Although the proliferation of undifferentiated preadipocytes plays an important part in hyperplasia, this process is less well understood than the post-proliferation differentiation process. Despite the potential importance of porcine visceral adipose tissue to both meat production and biomedical research, there has been little study of this tissue and, in particular, its development and differentiation. To detect the genes involved in the maintenance of porcine visceral preadipocytes in an undifferentiated state or in the inhibition of adipocyte differentiation, we performed suppression subtractive hybridization using mesenteric preadipocytes in which fragments of the genes that are downregulated at 2 d of differentiation were enriched. We selected 672 clones and subjected them to differential screening and semiquantitative reverse transcription (RT)-PCR. As a result, we identified 34 downregulated genes. Among these, the detailed expression patterns of 6 genes were examined using real-time RT-PCR in both preadipocytes during in vitro differentiation and cell fractions directly isolated from pig mesenteric adipose tissue. The expressions of connective tissue growth factor, AXL receptor tyrosine kinase, stromal membrane-associated protein l-like, and retinoic acid-induced 14 were significantly downregulated during adipocyte differentiation in vitro (P < 0.05), and the expressions of Rho/Rac guanine nucleotide exchange factor 2 and secreted frizzled-related protein 4 also tended to be decreased, although not significantly. Furthermore, all 6 genes showed significantly greater expression in stromal vascular cells, which contain preadipocytes, than in mature adipocytes (P < 0.05), raising the possibility that these genes are involved in adipocyte differentiation in vivo as well as in vitro. Key words: differentiation, gene expression, mesenteric adipocyte, mesenteric adipose tissue, porcine, suppression subtractive hybridization

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&apos;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 [...]

NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a twofold increase in glucose uptake in parallel with the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their total expression levels. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. The stimulatory effect of NCX 4016 on glucose uptake appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP cyclase/ protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin-signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the mitogen-activated protein kinases p38 and c-Jun N[H.sub.2]-terminal kinase and no activation of other stress-activated signaling molecules, including extracellular signal-regulated kinase, inhibitory factor [kappa]B, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocytes through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for the treatment of patients suffering from metabolic syndrome and type 2 diabetes. nitric oxide donation; adipocytes; glucose transporter translocation; S-nitrosylation; diabetes

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 Elovl3 gene, which putatively encodes for a protein involved in the elongation of saturated and monounsaturated fatty acids in the C20-C24 range, is expressed in murine liver, skin, and brown adipose tissue (BAT). In BAT, Elovl3 is dramatically upregulated during tissue activation in response to cold exposure, and functional data imply that ELOVL3 is a critical enzyme for lipid accumulation in brown adipocytes during the early phase of tissue recruitment. The activation of BAT is controlled by sympathetic nerve activity and norepinephrine release. By using primary cultures of brown adipocytes, we show here that the induced Elovl3 gene expression is synergistically regulated by norepinephrine and the peroxisome proliferator-activated receptor (PPAR) [gamma] ligand rosiglitazone. In addition, the potency of rosiglitazone to induce Elovl3 expression was several orders of magnitude higher than for the PPAR[alpha] and PPAR[delta] ligands WY-14643 and L-165041, respectively. The maximal increase in mRNA level by norepinephrine and rosiglitazone is achieved by induced transcription as well as increased mRNA stability, and the whole process requires novel protein synthesis. We conclude that norepinehrine and PPAR[gamma], despite having different roles in brown adipocyte activation and differentiation, cooperate in expanding the intracellular lipid pool by synergistically stimulating Elovl3 expression. fatty acid synthesis; fatty acid elongation; very long-chain fatty acids; lipid metabolism; peroxisome proliferator-activated receptor-[gamma]

Both monocyte chemoattractant protein-1 (MCP-1), a member of chemokine family, and angiotensinogen, a precursor of angiotensin (ANG) II, are produced by adipose tissue and increased in obese state. MCP-1 has been shown to decrease insulin-stimulated glucose uptake and several adipogenic genes expression in adipocytes in vitro, suggesting its pathophysiological significance in obesity. However, the pathophysiological interaction between MCP-1 and ANG II in adipose tissue remains unknown. The present study was undertaken to investigate the potential mechanisms by which ANG II affects MCP-1 gene expression in rat primary cultured preadipocytes and adipose tissue in vivo. ANG II significantly increased steady-state MCP-1 mRNA levels in a time-and dose-dependent manner. The ANG II-induced MCP-1 mRNA and protein expression was completely abolished by ANG II type 1 (AT1)-receptor antagonist (valsartan). An antioxidant/NF-[kappa]B inhibitor (pyrrolidine dithiocarbamate) and an inhibitor of 1 [kappa]B-[alpha] phosphorylation (Bay 11-7085) also blocked ANG II-induced MCP-1 mRNA expression. ANG II induced translocation of NF-[kappa]B p65 subunit from cytoplasm to nucleus by immunocytochemical study. Luciferase assay using reporter constructs containing MCP-1 promoter region revealed that two NF-[kappa]B binding sites in its enhancer region were essential for the ANG II-induced promoter activities. Furthermore, basal mRNA and protein of MCP-1 during preadipocyte differentiation were significantly greater in preadipocytes than in differentiated adipocytes, whose effect was more pronounced in the presence of ANG II. Exogenous administration of ANG II to rats led to increased MCP-1 expression in epididymal, subcutaneous, and mesenteric adipose tissue. In conclusion, our present study demonstrates that ANG II increases MCP-1 gene expression via ANG II type 1 receptor-mediated and NF-[kappa]B-dependent pathway in rat preadipocytes as well as adipose MCP-1 expression in vivo. Thus the augmented MCP-1 expression by ANG II in preadipocytes may provide a new link between obesity and cardiovascular disease. adipocyte; obesity

The expression profile of a series of adipokine genes linked to inflammation has been examined by quantitative PCR during the differentiation of human preadipocytes to adipocytes in primary culture, together with the integrated effects of TNF-[alpha] on the expression of these adipokines in the differentiated adipocytes. Expression of the genes encoding adiponectin, leptin, and haptoglobin was highly differentiation dependent, the mRNA being undetectable predifferentiation with the level peaking 9-15 days postdifferentiation. Although angiotensinogen (AGT) and monocyte chemoattractant protein-1 (MCP-1) were both expressed before differentiation, the mRNA level increased markedly on differentiation. The expression of nerve growth factor (NGF) and plasminogen activator inhibitor-1 (PAI-1) fell after differentiation, whereas that of TNF-[alpha] and IL-6 changed little. Measurement of adiponectin, leptin, MCP-1, and NGF in the medium by ELISA showed that the protein secretion pattern paralleled cellular mRNA levels. Treatment of differentiated human adipocytes with TNF-[alpha] (5 or 100 ng/ml for 24 h) significantly decreased the level of adiponectin, AGT, and haptoglobin mRNA (by 2- to 4-fold), whereas that of leptin and PAI-1 was unchanged. In contrast, TNF-[alpha] induced substantial increases in IL-6, TNF-[alpha], metallothionein, MCP-1, and NGF mRNAs, the largest increase being with MCP-1 (14.5-fold). MCP-1 and NGF secretion increased 8- to 10-fold with TNF-[alpha], whereas leptin and adiponectin did not change. These results demonstrate that there are major quantitative changes in adipokine gene expression during differentiation of human adipocytes and that TNF-[alpha] has a pleiotropic effect on inflammation-related adipokine production, the synthesis of MCP-1 and NGF being highly induced by the cytokine. nerve growth factor; tumor necrosis factor-[alpha]; white adipose tissue

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.

The effects of sex, genotype, and adipose depot on lipogenic enzyme activity have been investigated in Holstein and Pirenaican bulls and heifers, taking into account differences in adipocyte size. Fifteen Pirenaican bulls and 15 heifers and 15 Holstein bulls and 13 heifers were fattened until slaughter (12 to 13 mo old and 450 to 500 kg of body weight). During the fattening period, animals had ad libitum access to commercial concentrates and straw. The 10th rib was dissected to determine the fat content. Adipocyte size and activities of the following lipogenic enzymes were determined: glycerol 3-phosphate dehydrogenase, fatty acid synthase, nicotinamide adenine dinucleotide phosphate (NADP)-malate dehydrogenase, glucose 6-phosphate dehydrogenase, and NADP-isocitrate dehydrogenase, in the omental, perirenal, subcutaneous, and intermuscular adipose depots, respectively. Because adipocyte mean cell volume varied with sex, breed, and depot, regression analyses of [log.sub.e] activity per cell and [log.sub.e] cell volume were used to compare activities per unit volume. Sex, breed and depot had no effect (P > 0.05) on the gradients of regressions, which did not differ significantly from 1. Thus, activity per unit volume did not vary with cell size. Consequently, sex, breed, and depot effects on the regression analyses were equivalent to effects on activity per unit volume. Females had greater amounts of fat in the 10 th rib (P < 0.001), larger adipocytes (P < 0.001) and, in general, greater (P < 0.05) lipogenic activity per cell, even when adjusted for cell size, than males. These findings suggest that differences in adiposity between sexes are mainly due to females having a greater capacity for lipid synthesis, and hence, hypertrophy, than males. When adjusted for differences in carcass weight, Holsteins had larger adipocytes than Pirenaicans. The abdominal depots, omental and perirenal, had a greater adipocyte size (P < 0.001) and, in general, greater lipogenic enzyme activities per cell (P < 0.05) than the subcutaneous and intermuscular carcass depots. However, when activity per cell was adjusted for cell size, subcutaneous depots had greater fatty acid synthae, glucose 6-phosphate dehydrogenase, and NADP-malate dehydrogenase activities than omental and perirenal, indicating that other factors such as nutrient supply may restrict hypertrophy of carcass adipocytes. Key Words: Adipocytes, Adipose Tissue, Cattle, Genotypes, Lipogenesis, Sex

A QTL analysis of fat androstenone levels from a three-generation experimental cross between Large White and Meishan pig breeds was carried out. A total of 485 [F.sub.2] males grouped in 24 full-sib families, their 29 parents and 12 grandparents were typed for 137 markers distributed over the entire porcine genome. The [F.sub.2] male population was measured for fat androstenone levels at 100, 120, 140, and 160 d of age and at slaughter around 80 kg liveweight. Statistical analyses were performed using two interval mapping methods: a line-cross (LC) regression method, which assumes alternative alleles are fixed in founder lines, and a half- full-sib (HFS) maximum likelihood method, where allele substitution effects were estimated within each half- and full-sib family. Both methods revealed genomewide significant gene effects on chromosomes 3, 7, and 14. The QTL explained, respectively, 7 to 11%, 11 to 15%, and 6 to 8% of phenotypic variance. Three additional significant QTL explaining 4 to 7% of variance were detected on chromosomes 4 and 9 using LC method and on chromosome 6 using HFS method. Suggestive QTL were also obtained on chromosomes 2, 10, 11, 13, and 18. Meishan alleles were associated with higher androstenone levels, except on chromosomes 7, 10, and 13, although 10 and 13 additive effects were near zero. The QTL had essentially additive effects, except on chromosomes 4, 10, and 13. No evidence of linked QTL or imprinting effects on androstenone concentration could be found across the entire porcine genome. The steroid chromosome P450 21-hydroxylase (CYP21) and cytochrome P450 cholesterol side chain cleavage subfamily XIA (CYP11A) loci were investigated as possible candidate genes for the chromosome 7 QTL. No mutation of coding sequence has been found for CYP21. Involvement of a candidate regulatory mutation of CYP11A gene proposed by others can be excluded in our animals. Key Words: Androstenone, Boar Taint, Gene Mapping, Pigs, Quantitative Trait Loci

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.

A genetic mutation which promotes the growth of fat cells may be associated with obesity in some people. Researchers in Germany examined 358 people for mutations in the gene for the peroxisome-proliferator-activated receptor gamma 2, and found the mutant gene in four of 121 obese people. Only obese patients had the mutation, and those with it were considerably more overweight than obese people without the gene.

A study on the backbone and side chain mechanisms of uncomplexed human adipocyte lipid-binding protein (apo-A-LBP) and muscle fatty acid-binding proteins (apo-M-FABP) is presented. A-LBPs and M-FABPs are intracellular lipid-binding proteins that transport and store lipids. A-LBPs are found in fat cells while M-FABPs are found in skeletal muscles, the heart and the kidneys. These proteins have the same fold structure made up of 10 antiparallel beta strands and a helix-loop-helix design joining the A and B beta strands.

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]].

Experiments using mature adipocytes and preadipocytes from genetically obese Zucker rats show that both mature fat cells and newly differentiated adipocytes from obese rats are capable of maintaining genotypic differences even after long-term removal from their in vivo environment. Obese cells cultured in the absence of insulin and neurotransmitters exhibit higher fat storage capacity in adipocytes. This suggests the intrinsic expression of fatty mutation in prolonged, cultured mature adipocytes and in newly differentiated adipocytes.

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.

The HEK293 cells expressing endogenous AdipoR1/R2 is employed to understand the signaling mechanism and insulin-sensitizing effects in liver and skeletal muscle in adiponectin, an adipocyte-derived cytokine. The results demonstrated that adiponectin regulates Ras-dependent signaling and continuous exposure to adiponectin is sufficient to support serum-independent cell growth.

A study is conducted to show that differentiation of preadipocytes results in a concomitant decrease in the levels of Kruppel-like transcription factor 2 (KLF2) protein and also significantly reduces KLF2 promoter activity. The studies demonstrate that KLF2 does not affect the commitment of multipotent stem cells into the preadipocytic lineage but rather maintains their preadipocyte state and thereby negatively regulates their transition into adipocytes.

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.