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.     

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.     

Differential expression of plasminogen activator inhibitor-1, tumor necrosis factor-alpha,TNF-alpha converting enzyme and ADAMTS family members in murine fat territories

Our objective was to investigate expression of A disintegrin and metalloproteinase (ADAM) and ADAM proteins with a thrombospondin (TS) motif (ADAMTS) family members in adipose tissue of lean and obese mice. Five-week-old male mice were kept on standard chow (SFD) or on high fat diet (HFD) for 15 weeks, and subcutaneous (SC) and gonadal (GON) adipose tissue, as well as mature adipocytes and stromal-vascular (S-V) cells were harvested. mRNA levels of plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNF-alpha), ADAM-17 (TACE or TNF-alpha converting enzyme), ADAMTS-1 and ADAMTS-8 were quantified in isolated adipose tissues and cell fractions, and during differentiation of murine preadipocytes. The HFD resulted in a significantly enhanced weight of isolated SC and GON fat pads, and in enhanced blood levels of glucose, cholesterol and PAI-1. ADAM-17, TNF-alpha, PAI-1, ADAMTS-1 and ADAMTS-8 mRNA were detected in both SC and GON adipose tissue of lean mice (SFD). In SC adipose tissue of obese mice (HFD), the expression of ADAM-17 and PAI-1 was enhanced and that of ADAMTS-1 reduced, whereas in GON adipose tissue expression of TNF-alpha was enhanced and that of ADAMTS-8 reduced. In lean and obese mice, expression of ADAM-17, ADAMTS-1 and ADAMTS-8 was higher in the S-V cell fraction than in mature adipocytes. During differentiation of murine 3T3-F442A preadipocytes, expression of ADAM-17 and ADAMTS-1 remained virtually unaltered, whereas that of ADAMTS-8 decreased as adipocytes matured. Several ADAM and ADAMTS family members are expressed in adipose tissue and during differentiation of preadipocytes. Modulation of their expression upon development of obesity is adipose tissue-dependent.     

Genomic sequences capable of committing mouse and rat fibroblasts to adipogenesis.

The mouse Swiss 3T3-F442A/3T3-C2 cell system is well suited for the isolation of genes involved in commitment to adipogenesis. 3T3-F442A cells convert to adipocytes with high efficiency in response to confluence and insulin. The sister clonal line 3T3-C2 does not respond to these signals, but can convert to adipocytes when transfected with DNA from 3T3-F442A preadipocytes or from human fat. Human fat-tissue biopsy FO46 DNA transfected into 3T3-C2 gave rise to fat foci after two rounds of transfection and selection. A cosmid library of a subclone of secondary transfectant 3T3-C2/FO46-1 was screened for the human repetitive Alu sequence. Five out of eight Alu+ recombinant clones committed 3T3-C2 cells to adipogenesis. The adipose commitment (AC) activity of one cosmid, p18A4, was found to reside in two small, non-identical, subcloned sequences 1.2kb and 2.0kb in length, each separately able to commit 3T3-C2, precrisis mouse and rat fibroblasts and the multipotential C3H10T1/2 cell line to adipogenesis. We conclude that commitment to adipogenesis can be effected in vitro with high efficiency by transfection of specific sequences into a variety of host cells.     

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.     

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.     

Coculture of endothelial cells and mature adipocytes actively promotes immature preadipocyte development in vitro

Adipose tissue consists of mature adipocytes and endothelial cells, which are all supported by the extracellular matrix. Adipose tissue development is closely associated with angiogenesis. However, the adipocyte-endothelial cell interaction is unclear. To address this issue, we examined the effects of endothelial cells on the growth, apoptosis, and differentiation of mature adipocytes in three-dimensional collagen gel culture of the adipocytes with or without rat lung endothelial (RLE) cells. Spindle-shaped preadipocytes, an immature type of adipocyte, developed more actively around the adhesion sites of RLE cells to mature adipocytes in the coculture (rate of preadipocytes: 18.9+/-4.3%) than in the culture of adipocytes alone (2.0+/-5.1%). With respect to growth, RLE cells induced about a three-fold increase in bromodeoxyuridine uptake of mature adipocytes alone, while RLE cells did not influence the uptake of preadipocytes. RLE cells also did not affect the apoptotic indices by immunohistochemistry for single-stranded DNA in mature adipocytes or preadipocytes. These phenomena were not reproduced by RLE cell-conditioned medium, or by certain endothelial cell-produced cytokines. Our in vitro study is the first demonstration that endothelial RLE cells promote the active development of preadipocytes together with increased growth of mature adipocytes. These results suggest that endothelial cells are involved in the enlargement mechanism of adipose tissue mass through their direct adhesion to mature adipocytes.     

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.     

Thyroid hormone stimulates adipocyte differentiation of 3T3 cells

Triiodothyronine added at 0.1 nM to 3T3-F442A cells cultured in adipogenic medium having endogenous hormone concentrations similar to those of hypothyroid serum stimulated adipose conversion; activities of both lipogenic enzymes, glycerophosphate dehydrogenase and malic enzyme, increased with hormone treatment. The number of adipocytes was also augmented by L-T3 addition but the number of fat cell clusters remained the same as compared to non-treated cultures, suggesting that thyroid hormone increased the number of adipocytes probably through stimulating selective multiplication of precursor adipose cells. Hormone addition to cells cultured with non-adipogenic medium did not promote conversion showing that L-T3 is not an adipogenic factor by itself. Triiodothyronine added at concentrations similar to those found in hyperthyroidism, from 10 nM up to 10 µM, also increased the proportion of adipocytes without changing the number of fat cell clusters, but they decreased the activity of both lipogenic enzymes and lipid accumulation in mature adipocytes. It can be concluded that during 3T3-F442A differentiation into adipocytes L-T3 increases the number of differentiated adipocytes and, at low concentrations, also enhances lipogenic enzyme activities, whereas at the hyperthyroid hormone levels these enzyme activities are significantly reduced, remaining at levels similar to those of cells cultured with hypothyroid medium. This cloned cell line seems to be a useful model to study thyroid hormone action at both molecular and cellular level.     

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.     

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.     

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.     

Hormone-sensitive adenylyl cyclase in preadipocytes cultured from adipose tissue: comparison with 3T3-L1 cells and adipocytes

The adenylyl cyclase system of preadipocytes derived from the stromal vascular fraction of perirenal rat fat pads was characterized. Unlike mature adipocytes, preadipocyte adenylyl cyclase was only weakly stimulated by catecholamines and adrenocorticotrophic hormone, but was stimulated by guanine nucleotides. Parathyroid hormone and 2-chloroadenosine also stimulated preadipocyte adenylyl cyclase. The adenylyl cyclase system of preadipocytes resembled that of undifferentiated 3T3-L1 cells. However, agents which induced the differentiation of the 3T3-L1 cell adenylyl cyclase system did not have a similar effect on preadipocytes. A medium (CDM6) which induced some differentiation of preadipocyte adenylyl cyclase was developed. The observations that the adenylyl cyclase system of preadipocytes and undifferentiated 3T3-L1 cells are similar, that preadipocyte adenylyl cyclase can be induced to develop along lines similar to early differentiation of 3T3-L1 cells, and that the adenylyl cyclase system of fully-differentiated 3T3-L1 cells has characteristics intermediate between preadipocytes and adipocytes, suggest that the differentiation of preadipocyte and 3T3-L1 adenyly cyclase in vitro mimics adipose adenylyl cyclase development in vivo. The increased catecholamine and ACTH stimulation, and reduced GTP and adenosine sensitivities of adipocytes compared to preadipocytes suggest that a number of genes affecting adenylyl cyclase-associated regulatory and receptor proteins are coordinately repressed and derepressed during development.     

Impaired fat storage capacity in adipocyte precursors of I versus C57BL mice

I mouse strain displays adipocyte hypoplasia responsible for smaller fat pad size compared with C57BL mice. We investigated possible alterations in the proliferation and/or differentiation capacity of preadipocytes from the stroma-vascular fraction of adipose tissue in the I mouse strain. Control C57BL and I mice were studied at 8 weeks of age, and both adipose and stromal cells were isolated from epididymal and inguinal adipose tissue localizations. Results showed that the lower epididymal adipose mass in I mice was accompanied by a decrease in stromal cell number compared with C57BL mice. In inguinal fat pads, total cell number in the stroma-vascular fraction was unmodified; lipoprotein lipase activity significantly increased in stromal cells from I mice compared with control mice. In this depot, further characterization of cells from the stroma-vascular fraction by separation of cells according to density showed an increased number of preadipocytes in the I mouse whole stromal cell population. These preadipocytes seemed unable to undergo terminal maturation, thus leading to a decrease in the number of mature adipocytes. These results indicated that resistance to fat accumulation in I mice is characterized by site-dependent impairment of both the proliferative rate and the differentiation capacity of adipocyte precursors.     

Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue

We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro. This work was supported by grants from the Japan Ministry of Education, Science, Sports, and Culture (no. 19580348) and from MEXT. HAITEKU (2007–2011).