Secretion of a lysophospholipase D activity by adipocytes: involvement in lysophosphatidic acid synthesis

The aim of the present work was to depict the metabolic pathways involved in extracellular production of lysophosphatidic acid (LPA) by adipocytes. LPA was followed by quantifying the accumulation of LPA in the incubation medium (conditioned medium, CM) of 3T3F442A adipocytes or human adipose tissue explants using a radioenzymatic assay. Surprisingly, after separation from the cells, the amount of LPA present in CM could be significantly increased by further incubation at 37 degrees C. This suggested the presence of a LPA-synthesizing activity (LPA-SA) in CM. LPA-SA appeared as a soluble activity which was inhibited by divalent ion chelators EDTA and phenanthrolin. The effect of EDTA was preferentially reverted by CoCl2, as described for a lysophospholipase D (lyso-PLD) activity previously identified in rat plasma. LPA concentration could also be increased by treatment with a bacterial PLD, demonstrating the presence of PLD-sensitive LPA precursors (mainly lysophosphatidylcholine) in adipocyte CM. LPA-SA could be increased by the addition of exogenous lysophosphatidylcholine, lysophosphatidylglycerol, or lyso-platelet activating factor, demonstrating that LPA-SA resulted from the action of a lyso-PLD. LPA-SA was not inhibited, but rather activated, by primary alcohol (ethanol and 1-butanol), suggesting that adipocyte lyso-PLD was not a classical PLD. Finally, LPA-SA was found to be weaker in CM of undifferentiated adipocyte (preadipocytes) compared with CM of differentiated adipocytes. In conclusion, our results reveal the existence of a secreted lyso-PLD activity regulated during adipocyte-differentiation and involved in extra cellular production of synthesis of LPA by adipocytes.     

Autotaxin is released from adipocytes,catalyzes lysophosphatidic acid synthesis,and activates preadipocyte proliferation. Up-regulated expression with adipocyte differentiation and obesity

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904-910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipase d-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabetic db/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.     

A central role for hepatocyte growth factor in adipose tissue angiogenesis

Hepatocyte growth factor (HGF) is a potent mitogenic and angiogenic factor produced in human adipose tissue. In this study, we use 3T3-F442A preadipocytes to study the contribution of HGF to angiogenesis in an in vivo fat pad development model. As observed for human adipocytes, HGF is synthesized and secreted by 3T3-F442A preadipocytes and mature adipocytes. HGF knockdown with small-interfering RNA reduced HGF mRNA expression 82.3 +/- 4.2% and protein secretion 82.9 +/- 1.4% from 3T3-F442A preadipocytes. Silencing of HGF resulted in a 70.5 +/- 19.0% reduction in endothelial progenitor cell migration to 3T3-F442A-conditioned medium in vitro. 3T3-F442A preadipocytes injected under the skin of mice form a fat pad containing mature, lipid-filled adipocytes and a functional vasculature. At 72 h postinjection, expression of the endothelial cell genes TIE-1 and platelet endothelial cell adhesion molecule (PECAM)-1 was decreased 94.4 +/- 2.2 and 91.5 +/- 2.5%, respectively, in 3T3-F442A fat pads with HGF silencing. Knockdown of HGF had no effect on differentiation of 3T3-F442A preadipocytes to mature adipocytes in vitro or in vivo. In developing fat pads under the skin of HGF overexpressing transgenic mice, TIE-1 and PECAM-1 mRNA was increased 16.5- and 21.4-fold, respectively, at 72 h postinjection. The increase in gene expression correlated with immunohistochemical evidence of endothelial cell migration in the developing fat pad. These data suggest that HGF has a central role in regulating angiogenesis in adipose tissue.     

Dexamethasone-dependent expression of beta 1-24 corticotropin stimulated adenylate cyclase during adipose conversion of 3T3-F442A cells

When 3T3-F442A preadipocytes were grown in culture media supplemented with corticosteroid poor fetal calf serum and insulin they differentiated into adipocytes. Glycerophosphate dehydrogenase, a marker of terminal differentiation, developed a 600-fold increase of activity whereas the adenylate cyclase system remained unresponsive to the synthetic ACTH(1-24) analog. In contrast, 3T3-F442A adipocytes, differentiated in the presence of dexamethasone, exhibited an adenylate cyclase activity which was stimulated 4-fold by ACTH(1-24). The stimulation of the adenylate cyclase activity by GTP gamma S remained unchanged (about 20-25-fold) suggesting that the G regulatory coupling protein was not functionally modified by dexamethasone. Binding studies with 125I-ACTH revealed that specific cellular binding could be evidenced in dexamethasone-treated cells while control adipocytes did not exhibit any specific binding of 125I-ACTH. These findings lend support to the hypothesis that the setting off of this ACTH responsiveness in 3T3-F442A cells is regulated by dexamethasone after cells are committed to adipose differentiation.     

Aortic carboxypeptidase-like protein is regulated by transforming growth factor beta in 3T3-L1 preadipocytes

Adipogenesis is characterized by early remodeling of the extracellular matrix, allowing preadipocytes to adopt a more spherical shape and optimize lipid accumulation as they mature. Aortic carboxypeptidase-like protein (ACLP), found in collagen-rich tissues including adipose tissue, is expressed in 3T3-L1 and 3T3-F442A preadipocytes, and is downregulated during adipogenesis. We now report that ACLP is found in medium conditioned by 3T3-L1 preadipocytes. Transforming growth factor (TGF) beta, a known modulator of fibrillar matrix protein production, increased ACLP expression by 2.4+/-0.4-fold (mean+/-SE; n=3) in 3T3-L1 preadipocytes, through a mechanism that requires p42/44 MAPK activity. Addition of TGFbeta to differentiation medium, which inhibits adipogenesis, raised ACLP levels in 3T3-L1 cells. However, sustained expression of ACLP in stable clones of 3T3-L1 or 3T3-F442A preadipocytes did not interfere with adipogenesis.     

Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes

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.     

Decreased biosynthesis of actin and cellular fibronectin during adipose conversion of 3T3-F442A cells. Reorganization of the cytoarchitecture and extracellular matrix fibronectin

Differentiation of 3T3-F442A cells was accompanied by changes in cell morphology, decreased synthesis and assembly of actin and fibronectin. The network of microfilament stress fibers detected with NBD-phallacidin was altered during adipose conversion of 3T3-F442A cells. Parallel to this, the disappearance of fibrillar bundles of extracellular matrix fibronectin was observed by immunofluorescence staining. The pericellular fibronectin content, detected by immunoblotting, strongly diminished during the differentiation process. An altered rate of biosynthesis of both proteins was also measured by [35S]-methionine pulse-labeling and immunoprecipitation. A 4-5-fold decrease in cellular fibronectin synthesis was observed in adipocytes compared to control preadipocytes. Conversely, non-differentiating 3T3-C2 control cells did not reorganize either the cytoskeletal architecture or the extracellular matrix fibronectin in the resting state. These results suggest that the decreased rate of biosynthesis of cell-associated fibronectin is correlated with that of actin. Moreover, both events can essentially be ascribed to differentiation.     

Characterization of the long pentraxin PTX3 as a TNFalpha-induced secreted protein of adipose cells

Exposure of preadipocytes to long-chain fatty acids induces the expression of several markers of adipocyte differentiation. In an attempt to identify novel genes and proteins that are regulated by fatty acids in preadipocytes, we performed a substractive hybridization screening and identified PTX3, a protein of the pentraxin family. PTX3 mRNA expression is transient during adipocyte differentiation of clonal cell lines and is absent in fully differentiated cells. Stable overexpression of PTX3 in preadipocytes has no effect on adipocyte differentiation. In line with this, PTX3 mRNA is expressed in the stromal-vascular fraction of adipose tissue, but not in the adipocyte fraction; however, in 3T3-F442A adipocytes, the PTX3 gene can be reinduced by tumor necrosis factor alpha (TNFalpha) in a dose-dependent manner. This effect is accompanied by PTX3 protein secretion from both 3T3-F442A adipocytes and explants of mouse adipose tissue. PTX3 mRNA levels are found to be higher in adipose tissue of genetically obese mice versus control mice, consistent with their increased TNFalpha levels. In conclusion, PTX3 appears as a TNFalpha-induced protein that provides a new link between chronic low-level inflammatory state and obesity.     

Beta-Mecaptoethanol Suppresses Inflammation and Induces Adipogenic Differentiation in 3T3-F442A Murine Preadipocytes

Preadipocytes are present in adipose tissues throughout adult life that can proliferate and differentiate into mature adipocytes in response to environmental cues. Abnormal increase in adipocyte number or size leads to fat tissue expansion. However, it is now recognized that adipocyte hypertrophy is a greater risk factor for metabolic syndrome whereas fat tissue that continues to produce newer and smaller fat cells through preadipocyte differentiation is "metabolically healthy". Because adipocyte hypertrophy is often associated with increased oxidant stress and low grade inflammation, both are linked to disturbed cellular redox, we tested how preadipocyte differentiation may be regulated by beta-mercaptoethanol (BME), a pharmacological redox regulator and radical scavenger, using murine 3T3-F442A preadipocytes as the cell model. Effects of BME on adipogenesis were measured by microphotography, real-time PCR, and Western analysis. Our data demonstrated that preadipocyte differentiation could be regulated by extracellular BME. At an optimal concentration, BME enhanced expression of adipogenic gene markers and lipid accumulation. This effect was associated with BME-mediated down-regulation of inflammatory cytokine expression during early differentiation. BME also attenuated TNFalpha-induced activation of NFkappaB in differentiating preadipocytes and partially restored TNFalpha-mediated suppression on adipogenesis. Using a non-adipogenic HEK293 cell line transfected with luciferase reporter genes, we demonstrated that BME reduced basal and TNFalpha-induced NFkappaB activity and increased basal and ciglitazone-induced PPARgamma activity; both may contribute to the pro-adipogenic effect of BME in differentiating F442A preadipocytes.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

Proteomic analysis of adipocyte differentiation: Evidence that alpha2 macroglobulin is involved in the adipose conversion of 3T3 L1 preadipocytes

Adipogenesis is an important aspect of energy homeostasis. Here we have used a differential proteome mapping strategy to identify intracellular proteins that are differentially expressed during adipose conversion of 3T3 L1 preadipocytes. Two-dimensional gel electrophoresis analysis identified 8 proteins that are induced following hormone-evoked differentiation. In addition, we found that a alpha2 macroglobulin fragment was abundantly present in 3T3 L1 preadipocytes, but was virtually undetectable in fully differentiated adipocytes. Metabolic radiolabeling with (35S)methionine and Northern blot analysis indicated that the intracellular alpha2 macroglobulin fragment in preadipocytes was derived from the extracellular culture medium, not de novo synthesis. Incubation of preadipocytes with an antialpha2 macroglobulin polyclonal antibody caused depletion of the intracellular alpha2 macroglobulin fragments, and also enhanced spontaneous adipose conversion. These results suggest that intracellular alpha2 macroglobulin fragment inhibits adipocyte differentiation, and that hormone treatment induces differentiation at least in part by suppression of intracellular alpha2 macroglobulin activity in 3T3 L1 preadipocytes.     

Reconstituted basement membrane potentiates in vivo adipogenesis of 3T3-F442A cells

Adipocytes forming fat pad in vivo are surrounded by well developed basement membranes. Synthesis of basement membrane is enhanced during in vitro differentiation of preadipocyte line. In order to know the role of basement membrane in adipogenesis in vivo, we injected 3T3-F442A preadipocytes subcutaneously into nude mice together with or without the reconstituted basement membrane, Matrigel. Histological sections of the fat pads newly formed by injecting the cell alone showed dense population of immature adipocytes and microvessels within 2 weeks and they matured rapidly. In contrast, injection of the cells together with Matrigel showed sparse adipocytes after 2 weeks and they matured slowly over the period of 6 weeks. Quantification of the process by measuring the weight, DNA content, triglyceride content and glycerophosphate dehydrogenase (GPDH) activity of the fat pads showed that injection of the cell alone resulted in early maturation of adipose tissue with fewer adipocytes while the presence of Matrigel decelerated but potentiated the maturation of adipose tissue with 2 fold contents of DNA, triglyceride and GPDH activity. We thus showed that reconstituted basement membrane (Matrigel) supported the survival and maturation of adipocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient adenoviral transduction of 3T3-F442A preadipocytes without affecting adipocyte differentiation

The preadipocyte cell lines 3T3-L1 and 3T3-F442A are widely used to study the cellular mechanisms of preadipocyte differentiation and mature adipocyte functions. However, transfection with naked DNA is inefficient in these cell lines. Adenoviral gene transfer is a powerful technique to induce high levels of transgene expression. After failing to obtain 3T3-F442A stable transfectants, we studied different techniques designed to enhance the efficiency of adenoviral transduction in fat cells. First, we compared the effects of two agents known to significantly enhance adenoviral transgene transduction, namely the cationic lipid lipofectamine and the cationic polymer polylysine. We show here that lipofectamine-assisted adenoviral transduction was more efficient in 3T3-F442A than in 3T3-L1 preadipocytes at all tested multiplicity of infection. Lipofectamine, and more efficiently polylysine, yielded high and sustained levels of adenoviral transgene expression in 3T3-F442A preadipocytes. Adenoviral transgene expression was maintained throughout the differentiation process. Furthermore, the two agents also efficiently enhanced adenoviral transduction in mature 3T3-F442A adipocytes. Interestingly, neither protocol affected the differentiation process, morphological features or protein expression of mature adipocytes. These approaches could be of interest to study fat cell differentiation and the functions of mature adipocytes.     

Growth hormone (GH) differentially regulates NF-kB activity in preadipocytes and macrophages: implications for GH’s role in adipose tissue homeostasis in obesity

Adipose tissue remodeling in obesity involves macrophage infiltration and chronic inflammation. NF-kB-mediated chronic inflammation of the adipose tissue is directly implicated in obesity-associated insulin resistance. We have investigated the effect of growth hormone (GH) on NF-kB activity in preadipocytes (3T3-F442A) and macrophages (J774A.1). Our studies indicate that whereas GH increases NF-kB activity in preadipocytes, it decreases NF-kB activity in macrophages. This differential response of NF-kB activity to GH correlates with the GH-dependent expression of a cadre of NF-kB-activated cytokines in these two cell types. Activation of NF-kB by GH in preadipocytes heightens inflammatory response by stimulating production of multiple cytokines including TNF-α, IL-6, and MCP-1, the mediators of both local and systemic insulin resistance and chemokines that recruit macrophages. Our studies also suggest differential regulation of miR132 and SIRT1 expression as a mechanism underlying the observed variance in GH-dependent NF-kB activity and altered cytokine profile in preadipocytes and macrophages. These findings further our understanding of the complex actions of GH on adipocytes and insulin sensitivity.     

Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture

When cultured 3T3-F442A cells undergo adipose differentiation, they produce extracellular matrix (ECM) that is not present in undifferentiated cells. This ECM stains strongly with ruthenium red, tannic acid and with Alcian blue at both pH 1 and 2.5, showing histochemical characteristics similar to sulphated and non-sulphated glycosaminoglycans. Under the electron microscope, ECM was observed bound to the cell surface and in the intercellular space; it was composed of fibrils of several thicknesses with attached granules and fibrous long-spacing forms of collagen. In addition, adipocytes were observed as rounded cells interconnected with the ECM fibrils, thus giving rise to fat cell clusters similar to the adipocyte lobules found in adipose tissue. Since fat cell clusters in culture emerge by clonal expansion of one adipose precursor cell, we suggest that this ECM can keep daughter adipocytes interconnected during differentiation. ECM production by adipocytes might have some significance for the formation of fat cell lobules in vivo.