Development of a microscale cell culture analog to probe naphthalene toxicity

Prediction of human response to drugs or chemicals is difficult as a result of the complexity of living organisms. We describe an in vitro model that can realistically and inexpensively study the adsorption, distribution, metabolism, elimination, and potential toxicity (ADMET) of chemicals. A microscale cell culture analog (microCCA) is a physical replica of the physiologically based pharmacokinetics (PBPK) model. Such a microfabricated device consists of a fluidic network of channels to mimic the circulatory system and chambers containing cultured mammalian cells representing key functions of animal "organ" systems. This paper describes the application of a two-cell system, four-chamber microCCA ("lung"-"liver"-"other tissue"-"fat") device for proof-of-concept study using naphthalene as a model toxicant. Naphthalene is converted into reactive metabolites (i.e., 1,2-naphthalenediol and 1,2-naphthoquinone) in the "liver" compartment, which then circulate to the "lung" depleting glutathione (GSH) in lung cells. Such microfabricated in vitro devices are potential human surrogates for testing chemicals and pharmaceutics for toxicity and efficacy.     

Induction of cell death by photodynamic therapy with a new synthetic photosensitizer DH-I-180-3 in undifferentiated and differentiated 3T3-L1 cells

To examine the photodynamic therapy (PDT) effect on adipocytes, we investigated whether PDT using DH-I-180-3, a new synthetic lipophilic photosensitizer, induced cell death of both undifferentiated and differentiated 3T3-L1. 3T3-L1 pre-adipocytes were differentiated into adipocytes in the culture medium containing pantothenate, insulin, dexamethasone, isobutylmethylxanthine, and troglitazone. PDT was applied to both undifferentiated and differentiated 3T3-L1. Photosensitizer uptake in fat cells was determined by measuring its mean fluorescence intensity. DH-I-180-3 mediated effectively PDT-induced cell death of both pre-adipocytes and adipocytes. And the photosensitizer was accumulated more rapidly in 3T3-L1 adipocytes, compared with other cancer cell lines. These results demonstrate that PDT is a potent cell death inducer in pre-adipocytes and adipocytes. Thus, PDT with DH-I-180-3 may be applied for a new therapeutic modality for obesity treatment.     

The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors

Whole animal testing is an essential part in evaluating the toxicological and pharmacological profiles of chemicals and pharmaceuticals, but these experiments are expensive and cumbersome. A cell culture analog (CCA) system, when used in conjunction with a physiologically based pharmacokinetic (PBPK) model, provides an in vitro supplement to animal studies and the possibility of a human surrogate for predicting human response in clinical trials. A PBPK model mathematically simulates animal metabolism by modeling the absorption, distribution, metabolism, and elimination kinetics of a chemical in interconnected tissue compartments. A CCA uses mammalian cells cultured in interconnected chambers to physically represent the corresponding PBPK. These compartments are connected by recirculating tissue culture medium that acts as a blood surrogate. The purpose of this article is to describe the design and basic operation of the microscale manifestation of such a system. Microscale CCAs offer the potential for inexpensive, relatively high throughput evaluation of chemicals while minimizing demand for reagents and cells. Using microfabrication technology, a three-chamber ("lung"-"liver"-"other") microscale cell culture analog (microCCA) device was fabricated on a 1 in. (2.54 cm) square silicon chip. With a design flow rate of 1.76 microL/min, this microCCA device achieves approximate physiological liquid-to-cell ratio and hydrodynamic shear stress while replicating the liquid residence time parameters in the PBPK model. A dissolved oxygen sensor based on collision quenching of a fluorescent ruthenium complex by oxygen molecules was integrated into the system, demonstrating the potential to integrate real-time sensors into such devices.     

Lipid Droplets Characterization in Adipocyte Differentiated 3T3-L1 Cells: Size and Optical Density Distribution

The 3T3-L1 cell line, derived from 3T3 cells, is widely used in biological research on adipose tissue. 3T3-L1 cells have a fibroblast-like morphology, but, under appropriate conditions, they differentiate into an adipocyte-like phenotype. During the differentiation process, 3T3-L1 cells increase the synthesis of triglycerides and acquire the behavior of adipose cells. In particular, triglycerides accumulate in lipid droplets (LDs) embedded in the cytoplasm. The number and the size distribution of the LDs is often correlated with obesity and many other pathologies linked with fat accumulation. The integrated optical density (IOD) of the LDs is related with the amount of triglycerides in the droplets. The aim of this study is the attempt to characterize the size distribution and the IOD of the LDs in 3T3-L1 differentiated cells. The cells were differentiated into adipocytes for 5 days with a standard procedure, stained with Oil Red O and observed with an optical microscope. The diameter, area, optical density of the LDs were measured. We found an asymmetry of the kernel density distribution of the maximum Feret’s diameter of the LDs with a tail due to very large LDs. More information regarding the birth of the LDs could help in finding the best mathematical model in order to analyze fat accumulation in adipocytes.Key words: Lipid droplet, 3T3-L1, adipocyte, fat, triglyceride accumulation, integrated optical density     

Induction of S100 protein in 3T3-L1 cells during differentiation to adipocytes and its liberating by lipolytic hormones

When confluent cultures of cloned mouse 3T3-L1 cells were differentiated to adipocytes by three days of treatment with a combination of 0.5 microM dexamethasone and 0.5 mM 1-methyl-3-isobutylxanthine, the S100 protein content in the cells increased markedly, as determined by a sensitive immunoassay system. The S100 protein induced in the cell was the alpha alpha form (S100ao), which is the predominant form of S100 protein in mouse adipose tissue. The S100ao concentration in preadipocytes was about 1-3 ng/mg protein, while the concentration in differentiated adipocytes was 60-200 ng/mg protein. The immunoblotting test of the crude extract of adipocytes confirmed that the immunoreactive substance in the cells was the alpha subunit of S100 protein. The treatment with 1-methyl-3-isobutylxanthine or dexamethasone alone neither elicited the S100 protein induction nor triacylglycerols accumulation in the cells. The accumulation of triacyglycerols in the cells was always preceded by the induction of S100ao protein under conditions where the differentiation to adipocytes was elicited. The induction of S100ao protein and accumulation of triacylglycerols in the cells treated with dexamethasone and 1-methyl-3-isobutylxanthine were inhibited by the addition of antimicrotubular drugs, colchicine and vinblastine, but not by cytochalasin B, an antimicrofilament drug. S100ao protein in 3T3-L1 adipocytes was released by incubation with a lipolytic hormone, adrenocorticotropic hormone or catecholamines, in a cyclic-AMP-dependent manner as observed with rat epididymal fat pads [Biochim. Biophys. Acta (1986) 889, 84-90]. These results also suggest that S100 protein may participate in the function of adipocytes.     

Cyclic AMP effects on cell-to-cell junctional membrane permeability during adipocyte differentiation of 3T3-L1 fibroblasts

Mouse 3T3-L1 fibroblast cells, also know as preadipocytes, differentiate in vitro into adipocytes when treated with promoting agents and acquire numerous properties characteristic of mature fat cells. We studied junctional cell-to-cell communication by measuring the incidence of electrical coupling and transfer of carboxy- fluorescein among these cells. When 3T3-L1 cells were induced to differentiate into adipocytes, they lost virtually all cell-cell communication. Preadipocytes that remained nondifferentiated after the treatment maintained normal communication. Loss of communication in the adipocytes invariably coincided with appearance of lipid droplets and not with other phenotypic changes. In the differentiating cells, loss of cell-to-cell communication and lipid accumulation was prevented if dibutyryl cyclic AMP and caffeine were present in the culture medium. Addition of dibutyryl cyclic AMP and caffeine to already differentiated adipocytes resulted in loss of lipid and simultaneously improved junctional permeability. The results demonstrate that in the in vitro 3T3-L1 cell system, (a) cell-to-cell communication and lipid synthesis are intimately related during the adipose conversion and (b) cAMP affects the expression of the two phenotypes.     

Prediction of naphthalene bioaccumulation using an adipocyte cell line model

A long-term goal of this research is to develop an in vitro model to study the metabolism, distribution, and fate of chemicals or pharmaceuticals in animals and humans. An important component of such a system is an in vitro model to study bioaccumulation of specific chemicals in adipose tissue. Due to the difficulties in maintaining primary adipocytes in culture and conducting reproducible experiments, transformed adipocyte cell lines have been used as an alternative. In this paper, several rodent preadipocyte cell lines (3T3-L1, 3T3-F442A, and TA1 cells) that differentiate into adipocytes when exposed to the appropriate stimuli are tested as an investigative tool to study naphthalene accumulation. The in vitro model is tested by comparison of its performance to that of primary adipocytes. All the experimental evidence supports the hypothesis that naphthalene accumulation is primarily dependent on the level of intracellular lipid. Furthermore, the level of naphthalene bioaccumulation is linearly correlated with the amount of triglyceride content with the slope of 37.7 +/- 0.5 microg of naphthalene/(mg of triglyceride). Indomethacin/dexamethasone/insulin are shown to be more effective in promoting preadipocyte differentiation than methylisobutylxanthine/dexamethasone/insulin. Additionally, external factors, such as the presence of albumin and serum in the medium, affect the cellular naphthalene uptake by decreasing the amount of naphthalene transported into fat cells. Among the three cell lines tested, 3T3-L1 adipocytes accumulated the highest intracellular lipid and, hence, yielded the highest level of naphthalene accumulation. Its ability to accumulate naphthalene is comparable to that of primary adipocytes. The 3T3-L1 adipocyte model is appropriate for studying the bioaccumulation of xenobiotics that are aromatic hydrocarbons.     

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.     

Interleukin-15 stimulates adiponectin secretion by 3T3-L1 adipocytes: evidence for a skeletal muscle-to-fat signaling pathway

Interleukin-15 (IL-15) is a cytokine which is highly expressed in skeletal muscle tissue, and which has anabolic effects on skeletal muscle protein dynamics both in vivo and in vitro. Additionally, administration of IL-15 to rats and mice inhibits white adipose tissue deposition. To determine if the action of IL-15 on adipose tissue is direct, the capacity of cultured murine 3T3-L1 preadipocytes and adipocytes to respond to IL-15 was examined. IL-15 administration inhibited lipid accumulation in differentiating 3T3-L1 preadipocytes, and stimulated secretion of the adipocyte-specific hormone adiponectin by differentiated 3T3-L1 adipocytes. The latter observation constitutes the first report of a cytokine or growth factor which stimulates adiponectin production. IL-15 mRNA expression by cultured 3T3-L1 adipogenic cells and C2C12 murine skeletal myogenic cells was also examined. Quantitative real-time PCR indicated IL-15 mRNA was expressed by C2C12 skeletal myogenic cells, and was upregulated more than 10-fold in differentiated skeletal myotubes compared to undifferentiated myoblasts. In contrast, 3T3-L1 cells expressed little or no IL-15 mRNA at either the undifferentiated preadipocyte or differentiated adipocyte stages. These findings provide support for the hypothesis that IL-15 functions in a muscle-to-fat endocrine axis which modulates fat:lean body composition and insulin sensitivity.     

3T3-L1 adipocyte glucose transporter (HepG2 class): sequence and regulation of protein and mRNA expression by insulin, differentiation, and glucose starvation

A glucose transporter cDNA (GLUT) clone was isolated from mouse 3T3-L1 adipocytes and sequenced. The nucleotide and deduced amino acid sequences were, respectively, 95 and 99% homologous to those of the rat brain transporter. The mouse cDNA and a polyclonal antibody recognizing the corresponding in vitro translation product were used to compare changes in transporter mRNA and protein levels during differentiation, glucose starvation, and chronic insulin exposure of 3T3-L1 preadipocytes. The respective cellular content of transporter mRNA and protein were increased 6.6- and 7.8-fold during differentiation, and 3.8- and 2.5-fold from chronic insulin exposure of differentiated adipocytes. Glucose starvation increased transporter mRNA and protein levels 2.2- and 3.5-fold in undifferentiated preadipocytes and 1.8- and 3.1-fold in differentiated adipocytes. Starvation of undifferentiated cells completely converted the native transporter to an incompletely glycosylated form, while increasing basal transport rates 4.5-fold. Either full glycosylation is not required to produce a functionally active transporter, or starvation causes a unique predifferentiation induction of the normally absent "responsive" transporter. The changes in transporter protein expression elicited by differentiation were attributed primarily to increased rates of transporter synthesis, while the disproportionate changes in mRNA and protein expression from chronic insulin treatment and starvation suggested these conditions increase synthesis and decrease turnover rates in regulating transporter protein expression. Although chronic insulin exposure and glucose starvation each raised the expression of transporter protein greater than 3-fold and basal transport rates 2.5- to 4.5-fold, no significant increase in the insulin responsiveness of 3T3-L1 preadipocytes or differentiated adipocytes was observed. Thus, the changes in the transporter mRNA and protein expression observed in this study were most consistent with their being associated with the regulated expression of a basal or low level insulin-responsive transporter.     

Lipoxygenase inhibitors inhibit heparin-releasable lipoprotein lipase activity in 3T3-L1 adipocytes and enhance body fat reduction in mice by conjugated linoleic acid

The t10c12 isomer of conjugated linoleic acid (CLA) reduces lipid accumulation in adipocytes in part by inhibiting heparin-releasable lipoprotein lipase (LPL) activity. We now show that inhibitors of lipoxygenase (LOX) activity (2-[12-hydroxydodeca-5,10-diynyl]-3,5,6-trimethyl-p-benzoquinone; 5,8,11,14-eicosatetraynoic acid; salicylhydroxamic acid; indomethacin; nordihydroguaiaretic acid (NDGA)) produce a similar inhibitory effect on LPL activity in cultured 3T3-L1 mouse adipocytes. Additionally the LOX inhibitors had no effect on, or inhibited, lipolysis in this cell system (measured as glycerol release). Growing mice fed diet containing 0.1% NDGA for 4 weeks displayed 21% reduction in body fat, which was similar to 23% reduction in body fat produced by feeding diet containing a suboptimal amount of CLA (0.1%) for 4 weeks. Feeding diet containing both 0.1% NDGA and 0.1% CLA resulted in 51% reduction in body fat which was accompanied by significant increases in whole body water and protein. Aspirin, an inhibitor of cyclooxygenase 1 and 2, had no effect on LPL activity in 3T3-L1 adipocytes, did not affect body composition when fed to growing mice, and failed to influence the effects of CLA on LPL activity in 3T3-L1 cells or body composition in mice. These findings appear to provide new perspectives and insights into the relationships between CLA, eicosanoids, the control of lipid accumulation in adipocytes, and effects of CLA on the immune system.     

Restricted localization of the adipocyte/muscle glucose transporter species to a cell surface-derived vesicle fraction of 3T3-L1 adipocytes. Inhibited lateral mobility of integral plasma membrane proteins in newly inserted membrane areas of differentiated 3T3-L1 cells

The recent demonstration of a large cell surface-derived pool of insulin-sensitive glucose transporters, presumably concentrated in the microvilli of 3T3-L1 adipocytes, induced the assumption that in differentiated adipocytes, newly inserted plasma membrane areas may display restricted lateral mobility, thereby preventing diffusion of integral membrane proteins out of these areas into the adjoining plasma membrane. In order to test this assumption, the cell surface distributions of the two glucose transporter species expressed by 3T3-L1 cells were determined using specific antisera against the HepG2/erythrocyte transporter, GluT1, which is synthesized in both fibroblasts and adipocytes, and the adipocyte/muscle-specific transporter, GluT4, expressed for the first time 3-4 days after induction of adipose conversion. GluT1 was shown to be localized in the plasma membrane of both 3T3-L1 preadipocytes and adipocytes, whereas GluT4 was almost entirely restricted to the low density surface-derived vesicle (LDSV) fraction of 3T3-L1 adipocytes most likely consisting of microvilli-derived vesicles. In contrast to the minor portion of GluT4 found in the adipocyte plasma membrane fraction, equal amounts of the GluT1 protein were detected in both the plasma membrane and the LDSV fractions of adipocytes. Both transporter species were present in the microsomal and the LDSV fractions of adipocytes. The observed distribution of the two transporter species is in accordance with the postulated restriction of the lateral mobility in plasma membrane areas formed by newly inserted transgolgi vesicles of differentiated adipocytes.     

Effects of homocysteine on adipocyte differentiation and CD36 gene expression in 3T3-L1 adipocytes

The aim of this study was to investigate the effects of homocysteine (Hcy), a risk factor for cardiovascular diseases, hypertension, stroke and obesity, on expression of CD36 that regulates uptake of oxidized low-density lipoprotein (Ox-LDL) by adipocytes and differentiation of 3T3-L1 cells to adipocytes. Cell viability was determined using MTT assay, and density of triglycerides were measured with Oil Red O staining. The expression levels of CD36 were analyzed using SYBR green assay by quantitative RT-PCR. Our results showed that the addition of Hcy inhibited differentiation of 3T3-L1 preadipocytes in a dose-dependent manner without a significant cell toxicity (p < 0.05). Percentage CD36 gene expression increased in the Hcy treatment groups, but not statistically significantly (p > 0.05) compared to differentiated adipocytes. Hcy reduced adipocyte differentiation, but had no effect on the expression level of CD36 in vitro conditions. The effect of Hcy on uptake and clearance of Ox-LDL by adipose tissue now needs to be investigated in vivo.     

Lipoxygenase inhibitors inhibit heparin-releasable lipoprotein lipase activity in 3T3-L1 adipocytes and enhance body fat reduction in mice by conjugated linoleic acid.

The t10c12 isomer of conjugated linoleic acid (CLA) reduces lipid accumulation in adipocytes in part by inhibiting heparin-releasable lipoprotein lipase (LPL) activity. We now show that inhibitors of lipoxygenase (LOX) activity (2-[12-hydroxydodeca-5,10-diynyl]-3,5,6-trimethyl-p-benzoquinone; 5,8,11,14-eicosatetraynoic acid; salicylhydroxamic acid; indomethacin; nordihydroguaiaretic acid (NDGA)) produce a similar inhibitory effect on LPL activity in cultured 3T3-L1 mouse adipocytes. Additionally the LOX inhibitors had no effect on, or inhibited, lipolysis in this cell system (measured as glycerol release). Growing mice fed diet containing 0.1% NDGA for 4 weeks displayed 21% reduction in body fat, which was similar to 23% reduction in body fat produced by feeding diet containing a suboptimal amount of CLA (0.1%) for 4 weeks. Feeding diet containing both 0.1% NDGA and 0.1% CLA resulted in 51% reduction in body fat which was accompanied by significant increases in whole body water and protein. Aspirin, an inhibitor of cyclooxygenase 1 and 2, had no effect on LPL activity in 3T3-L1 adipocytes, did not affect body composition when fed to growing mice, and failed to influence the effects of CLA on LPL activity in 3T3-L1 cells or body composition in mice. These findings appear to provide new perspectives and insights into the relationships between CLA, eicosanoids, the control of lipid accumulation in adipocytes, and effects of CLA on the immune system.     

Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.     

Insulin and TNF alpha induce expression of the forkhead transcription factor gene Foxc2 in 3T3-L1 adipocytes via PI3K and ERK 1/2-dependent pathways

We have recently identified the winged helix/forkhead gene Foxc2 as a key regulator of adipocyte metabolism that counteracts obesity and diet-induced insulin resistance. This study was performed to elucidate the hormonal regulation of Foxc2 in adipocytes. We find that TNF alpha and insulin induce Foxc2 mRNA in differentiated 3T3-L1 cells with the kinetics of an immediate early response (1-2 h with 100 ng/ml insulin or 5 ng/ml TNF alpha). This induction is, in both cases, attenuated by the PI3K inhibitor wortmannin as well as the MAPK kinase inhibitor PD98059. Furthermore, we show that stimulation of 3T3-L1 adipocytes with phorbol-12-myristate-13-acetate or 8-(4-chlorophenyl)thio-cAMP induces the expression of Foxc2. Interestingly, we find that the basal level of Foxc2 mRNA is down-regulated whereas hormonal responsiveness increases during differentiation of 3T3-L1 from preadipocytes to adipocytes. At the protein level, immunoblots with Foxc2 antibody demonstrated an induction of Foxc2 by insulin and TNF alpha in nuclear extracts of 3T3-L1 adipocytes. EMSA of nuclear proteins from phorbol-12-myristate-13-acetate- and TNF alpha-treated 3T3-L1 adipocytes using a forkhead consensus oligonucleotide revealed specific binding of a Foxc2/DNA complex. In conclusion, our data suggest that insulin and TNF alpha regulate the expression of Foxc2 via a PI3K- and ERK 1/2-dependent pathway in 3T3-L1 adipocytes. Also, signaling pathways downstream of PKA and PKC induce the expression of Foxc2 mRNA.     

Formation and characterization of spontaneously formed heterokaryons between quail myoblasts and 3T3-L1 preadipocytes: correlation between differential plasticity and degree of differentiation

Skeletal muscle cells and adipose cells have a close relationship in developmental lineage. Our previous study has shown that the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells (preadipocytes) normally differentiated into myotubes, whereas the heterokaryons between myoblasts and differentiated 3T3-L1 cells (adipocytes) failed myogenic differentiation. These results suggest differences between preadipocytes and adipocytes. The purpose of this study was to clarify whether preadipocytes have flexibility in differentiation before terminal adipose differentiation. Presumptive quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and mouse 3T3-L1 cells (either preadipocytes or adipocytes) were co-cultured for 48 h under conditions allowing myogenic differentiation. On co-culture between myoblasts and undifferentiated 3T3-L1 cells, heterokaryotic myotubes formed spontaneously, but not on co-culture with differentiated 3T3-L1 cells. In addition, the heterokaryotic myotubes expressed mouse myogenin derived from the 3T3-L1 cell gene. Our previous study indicated that the fusion sensitivity of differentiating myoblasts change with decreasing cholesterol of the cell membrane during myoblast fusion. Thus we compared the level of membrane cholesterol between undifferentiated and differentiated 3T3-L1 cells. The result showed that the level of membrane cholesterol in 3T3-L1 cells increases during adipose differentiation. Corresponding to the increase in membrane cholesterol content, differentiated 3T3-L1 cells had lower sensitivity to HVJ (Sendai virus)-mediated cell fusion than undifferentiated 3T3-L1 cells. This study demonstrated that 3T3-L1 cells at an undifferentiated state have a capacity for spontaneous fusion with differentiating myoblasts following myogenic differentiation, and that the capacity is lost after terminal adipose 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.