Studies of lipoprotein lipase during the adipose conversion of 3T3 cells.

Lipoprotein lipase activity is negligible in exponentially growing 3T3-L1 cells and 3T3-F442A cells, but develops in both lines when they reach a confluent state and undergo adipose conversion. 3T3-C2 cells, which undergo adipose conversion with extremely low frequency, do not develop the enzyme. The lipase activity of 3T3-L1 and 3T3-F442A is greatly enhanced by insulin and increases 80–180 fold during the adipose conversion. The lipase has the following characteristics in common with lipoprotein lipase from adipose and other tissues: it is dependent upon serum, is inhibited by 0.5–1.0 M sodium chloride, is recovered from acetone powders, has an alkaline pH optimum and is released from the cells by heparin. Like the lipoprotein lipase of tissue adipose cells, the enzyme of 3T3-L1 decays in the presence of cycloheximide with a half-time of about 25 min at 37°C.The ability of 3T3-F442A and 3T3-L1 to take up triglyceride from the medium depends almost completely upon lipoprotein lipase. They incorporate the fatty acids of a large fraction of a triglyceride emulsion added to the medium, and this utilization is stimulated by heparin. Very little of the glycerol portion of the triglyceride is incorporated. 3T3-C2, which lacks lipoprotein lipase, utilizes very little of either the fatty acid or the glycerol portion of triglyceride.The relevance of external lipid or lipoprotein to both the adipose conversion and the appearance of lipoprotein lipase was tested using confluent cultures in medium depleted of these components. In the presence of serum whose lipoproteins have been removed by flotation, lines 3T3-F442A and 3T3-L1 undergo adipose conversion as completely as in the presence of untreated serum, and lipoprotein lipase activity appears at essentially the same rate. In medium whose serum supplement has been extracted with acetone:ethanol, 3T3-F442A cells undergo adipose conversion to nearly the same extent as in untreated serum, and develop nearly the same increase in lipoprotein lipase activity.Unless even very low concentrations of lipids or lipoprotein are saturating it can be concluded that the adipose conversion does not depend upon external lipids or lipoproteins for its induction; rather the differentiation program is built into the cell type and comes into operation when growth is arrested even in their absence. The source of fatty acids utilized for triglyceride synthesis, however, may be affected by the amount of lipid provided to the cells.     

Properties of growth hormone receptors in relation to the adipose conversion of 3T3 cells

Cultured preadipose 3T3 cells undergo a process of differentiation in which they convert to adipose cells. Growth hormone promotes this conversion. Since 3T3 sublines vary in their susceptibility to adipose conversion, it was of interest to examine the properties of the growth hormone receptors in relation to that susceptibility. It was found that preadipose 3T3-F442A cells, which are able to convert to adipose cells with high frequency, are able to bind about 10(4) growth hormone molecules per cell with Kd approximately 10(-9) M. After adipose conversion, no appreciable change in hormone binding was detected. The binding of growth hormone to 3T3-C2 cells (a line virtually insusceptible to adipose conversion) was indistinguishable from that to 3T3-F442A cells. Internalization and degradation of the hormone were also similar in the two cell lines. Susceptibility to adipose conversion is therefore not determined by the relative ability of the cells to bind or degrade the hormone, but must instead depend on some response, as yet unidentified, that follows binding of the hormone.     

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.     

Increase in fatty acid synthetase content of 3T3-L cells undergoing spontaneous and chemically induced differentiation to adipocytes.

3T3-L fibroblasts differentiate into adipose cells when maintained in a non-growing state. The specific activity of fatty acid synthetase of differentiated cells was 25--30-fold higher than that present in 3T3-L fibroblasts or in 3T3-C2 cells that possess an extremely low incidence of differentiation to adipocytes. The results of immunochemical analysis indicate that the increased specific activity of fatty acid synthetase in the differentiated cells is due to an increase in the cellular content of this enzyme. The rate of conversion of adipose cells was accelerated by brief exposure of confluent non-growing cultures of 3T3-L cells to 3-isobutyl-1-methylxanthine. This was accompanied by an increase in the specificity activity of fatty acid synthetase, which was also shown to be due to an increase in the cellular content of this enzyme. The continuous presence of 3-isobutyl-1-methylxanthine in the culture medium was not required to elicit the morphological and biochemical changes in 3T3-L cells that occurred many days after the removal of the inducer but earlier than the onset of spontanous differentiation.     

Effect of differentiation on the adenylate cyclase system of 3T3-C2 and 3T3-L1 cells. Determination of choleragen substrates in differentiating 3T3-L1 and nondifferentiating 3T3-C2 cells

3T3-L1 preadipocytes, when treated with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, differentiate into cells with the morphological and biochemical properties of adipocytes; the closely related 3T3-C2 cells, under identical conditions, exhibit a low frequency of adipocyte conversion. During differentiation, 3T3-L1 preadipocytes acquire an increased responsiveness to certain agonists (e.g. isoproterenol and adrenocorticotropic hormone) that influence lipolysis and lipogenesis through activation of adenylate cyclase, whereas 3T3-C2 cells do not. It has been suggested that changes in hormone responsiveness of 3T3-L1 cells during differentiation result from increased amounts of the guanyl nucleotide-binding protein of adenylate cyclase, as demonstrated by choleragen-catalyzed [32P]ADP ribosylation of 42 and 49-50-kilodalton particulate peptides. Particulate fractions from nondifferentiating 3T3-C2 cells, like those from 3T3-L1 cells, contained choleragen substrates of 42 and 46-47 (doublet) kilodaltons. Incubation of intact 3T3-L1 or 3T3-C2 cells with choleragen prior to preparation of particulate fractions prevented the subsequent in vitro choleragen-dependent [32P]ADP ribosylation of only these peptides. Increased incorporation of radioactivity into both the 42 and 46-47-kilodalton peptides was observed during differentiation of 3T3-L1 cells. However, a similar increase was also observed in nondifferentiating 3T3-C2 cells subjected to the differentiation protocol. Therefore, increased hormone responsiveness of 3T3-L1 adipocytes cannot be explained solely on the basis of increased labeling, and perhaps increased amounts, of the guanyl nucleotide-binding protein.     

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.     

Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells

Cultured mouse 3T3-F442A and 3T3-C2 fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). This phosphoprotein is similar but not identical to the dsRNA-dependent eukaryotic initiation factor-2 (eIF-2) alpha protein kinase (dsI), which regulates protein synthesis in rabbit reticulocytes. We have studied the relationship between cell growth and phosphorylation of the 67K protein (designated 3T3-dsRNA-dependent eIF-2 alpha kinase). A low level of dsRNA-dependent phosphorylation of 3T3-dsI was detectable in extracts prepared from cells not treated with IFN and grown at a low cell density. The phosphorylation of dsI and the phosphorylation of a 38K protein identified as the alpha-subunit (38K) of 3T3-eIF-2 (eIF-2 alpha) occurred concomitantly; the levels of these phosphorylations confluent and thereafter decreased markedly. Treatment of cells with IFN at all stages of growth resulted in an increase in phosphorylation of dsI. 3T3-F442A and 3T3-C2 fibroblasts were found to produce and secrete IFN at levels sufficient to induce an elevated dsI activity.     

Differentiation of 3T3-L1 fibroblasts to adipocytes. Loss of stimulation of uridine and 2-deoxy-D-glucose transport by PGF2 alpha in the course of differentiation of the 3T3-L1 cell line.

Insulin and prostaglandin F₂ (PGF_2α) stimulate undine and 2-deoxy-d-glucose transport by 3T3-L1 and 3T3-C2 cells. Maximal stimulation of both transport systems is achieved with 1.5 μM PGF_2α. Maximal stimulation of uridine transport is achieved with 34 nM (200 ng/ml) insulin. The basal (control) level of uridine uptake is lower in 3T3-L1 than in 3T3-C2 cells, while the basal level of 2-deoxy-d-glucose uptake is slightly higher in 3T3-L1 cells. The stimulatory effect of PGF_2α on both transport systems is abolished in the course of differentiation of 3T3-L1 cells to adipocytes. Removal of the inducers of differentiation (insulin plus indomethacin) from the medium of differentiated cells does not restore responsiveness of either transport system to PGF_2α. In contrast the stimulatory effect of insulin on either transport system is not abolished during differentiation. Treatment of the non-differentiating 3T3-C2 cell line with inducers of differentiation reduces the basal level of 2-deoxy-d-glucose transport by 70% and only slightly decreases the basal level of uridine transport without affecting the stimulatory effect of PGF_2α on either transport system.     

EID1-induces brown-like adipocyte traits in white 3T3-L1 pre-adipocytes

PPARγ and pRB play an important role in the development of adipose cells, and functional modification of these proteins may lead to beneficial changes in adipose cell physiology. In the present work, we show that over-expression of EID1 (E1A-like inhibitor of differentiation), an inhibitor of muscle cell differentiation, reduces PPARγ ligand-dependent transactivation and decreases triglyceride stores in pre-adipocytes (3T3-L1 cells). Additionally, we found that EID1 binds to pRB at the onset of adipocyte differentiation and may act to reduce pRB levels. Over-expression of EID1 in 3T3-L1 cells leads to increased expression of UCP1 and PGC-1α, both of which are involved in caloric dissipation and thermogenesis, in brown adipose tissue. These results indicate that EID1 is able to reduce fat accumulation in adipose cells and induce expression of brown fat genes in pre-adipocytes (3T3-L1 cells) normally destined to become white fat cells. The functional reduction of PPARγ and pRB mediated by EID1 in adipose cells may play an important role in insulin resistance and the metabolic syndrome.     

Differentiation of 3T3-L1 fibroblasts to adipocytes. Effect of insulin and indomethacin on the levels of insulin receptors

Differentiating (3T3-L1) and nondifferentiating (3T3-C2) fibroblastic cell lines possess two classes of insulin receptors, high affinity (KD = 1.0 to 3.7 X 10(-9) M) and low affinity (KD = 2.0 to 3.6 X 10(-8) M). Confluent cultures of 3T3-L1 cells induced to differentiate by insulin (1.74 x 10(-6) M) or indomethacin (1.25 x 10(-4) M) exhibit a 3-fold increase in the number of high affinity and low affinity receptors per cell or a 1.5- to 1.8-fold increase in the number of receptors per micron2 of surface area. In contrast, nondifferentiating 3T3-C2 cells treated with insulin or indomethacin lose almost completely the high affinity insulin receptors while retaining the same levels of low affinity receptors. The loss of high affinity receptors of the 3T3-C2 cells is accompanied by the disappearance of the stimulatory effect of insulin on the production of CO2 from glucose and on the uptake of aminoisobutyrate. The levels of high affinity insulin receptors appear to be regulated by different mechanisms in the differentiating (3T3-L1) and nondifferentiating (3T3-C2) cell lines. The mode of this regulation may have a bearing on the ability of a particular cell line to differentiate.     

Differentiation of 3T3-L1 fibroblasts to adipocytes : Loss of stimulation of uridine and 2-deoxy- -glucose transport by PGF2α in the course of differentiation of the 3T3-L1 cell line

Insulin and prostaglandin F₂ (PGF_2α) stimulate undine and 2-deoxy- -glucose transport by 3T3-L1 and 3T3-C2 cells. Maximal stimulation of both transport systems is achieved with 1.5 μM PGF_2α. Maximal stimulation of uridine transport is achieved with 34 nM (200 ng/ml) insulin. The basal (control) level of uridine uptake is lower in 3T3-L1 than in 3T3-C2 cells, while the basal level of 2-deoxy- -glucose uptake is slightly higher in 3T3-L1 cells. The stimulatory effect of PGF_2α on both transport systems is abolished in the course of differentiation of 3T3-L1 cells to adipocytes. Removal of the inducers of differentiation (insulin plus indomethacin) from the medium of differentiated cells does not restore responsiveness of either transport system to PGF_2α. In contrast the stimulatory effect of insulin on either transport system is not abolished during differentiation. Treatment of the non-differentiating 3T3-C2 cell line with inducers of differentiation reduces the basal level of 2-deoxy- -glucose transport by 70% and only slightly decreases the basal level of uridine transport without affecting the stimulatory effect of PGF_2α on either transport system.     

Dexamethasone regulation of terminal differentiation in 3T3-F442A preadipocyte cell line

The 3T3-F442A preadipocyte cell line was previously shown to possess specific glucocorticoid receptors whose number increased in the time course of differentiation. We have examined the effects of a three day dexamethasone treatment, added at confluence, on cells differentiated in the presence or absence of insulin. Triglyceride accumulation, polyamine content as well as glycerophosphate dehydrogenase and fatty acid synthetase activities were measured during the adipose conversion. We have also determined 2-deoxyglucose uptake in non-differentiated and differentiated cells. Dexamethasone was shown to decrease the adipose conversion by 3T3-F442A cells in the presence or absence of insulin. Intracellular spermidine content in differentiating cells was sensitive to dexamethasone and insulin in the same way as an enzymatic marker of terminal differentiation, glycerophosphate dehydrogenase. Dexamethasone decreases the 2 deoxyglucose uptake in non-differentiated and differentiated cells while insulin increases this uptake only in differentiated cells. This work shows that glucocorticoids inhibit adipocyte metabolism at distinct levels and suggests that these hormones might play an important role in the regulation of adipose tissue mass.Abbreviations DEX dexamethasone - FAS fatty acid synthetase - GPDH glycerophosphate dehydrogenase - MIX 1-methyl-3-isobutylxanthine     

Coupling of growth arrest and expression of early markers during adipose conversion of preadipocyte cell lines

After growth arrest at the entry of the S phase of the cell cycle, Ob1771 and 3T3-F442A cells, but not 3T3-C2 cells, accumulate lipoprotein lipase and pOb24 mRNA that are early markers of adipose conversion. Removal of the single- or double-thymidine block when cultured cells are present at low density leads first to DNA synthesis and growth resumption, then to a continuous proliferation and a rapid disappearance of these markers. By contrast, growth-arrested Ob1771 cells reinoculated at high density undergo a single round of cell division, maintain high levels of early marker(s) and acquire with time both glycerol-3-phosphate dehydrogenase and lipids. Thus, depending upon the conditions in culture, growth-arrested cells can undergo either a dedifferentiation leading to a loss of early markers or a terminal differentiation leading to the acquisition of late markers.     

Hormonal regulation of amino acid uptake by cultured 3T3-L1 adipocytes

Incubation of the adipocytes for 20 hours with insulin or with Bt2cAMP plus the theophylline stimulated adipocyte uptake of AIB and MeAIB but did not stimulate the uptake of glutamine or cycloleucine. MeAIB uptake by both 3T3-L1 preadipocytes and 3T3-C2 cells was relatively unresponsive to insulin. However, MeAIB uptake by 3T3-C2 cells was stimulated by treatment with Bt2cAMP plus theophylline. Incubation of 3T3 adipocytes for 60 min with insulin yielded maximal stimulation of 2-deoxyglucose uptake but no stimulation of the uptake of AIB, MeAIB or glutamine. Responsiveness of transport to Bt2cAMP does not appear to require adipocyte differentiation. By contrast, adipocyte differentiation may be required for the development of the insulin-responsive transport systems.     

Neutral and acid lipase of mouse 3T3 fibroblasts with increased adipose conversion.

In the resting state, 3T3-L1 fibroblasts become adipose converted and increase their fatty acid and triglyceride synthetase. We have found that they contain four times the neutral lipase activity and 1.5 times the acid lipase activity of logarithmically dividing cells. The activities of lysosomal acid beta-galactosidase and N-acetyl-beta-D-glucosaminidase were the same in the adipose converted and logarithmically dividing cells. The data suggest a possible relation between the increased neutral lipase activity in 3T3-L1 cells and their adipose conversion and demonstrates that the adipose converted 3T3-L1 fibroblasts, unlike true adipose cells, contain high levels of lysosomal acid hydrolases.     

Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells

When their growth is arrested in culture, susceptible 3T3 fibroblasts differentiate into adipose cells. Different clones form adipose cells with different frequency, depending upon the proportion of susceptible cells they contain. In cultures grown from small inocula, the fat cells appear in clusters formed by colonies of susceptible cells. Study of these clusters indicates the infrequent occurrence of cellular transitions from insusceptible to susceptible state.Beginning with a clone converting to adipose cells with a very low frequency, it has been possible, by serial selection, to generate subclones which convert with a high frequency. This evolution is due to spontaneous heritable changes affecting susceptibility to the adipose conversion. Presumably, they involve the control of triglyceride synthesis.Early stages of the adipose conversion may be recognized in stained cultures. When triglyceride first begins to accumulate, the highly extended and flattened processes of the cells are probably similar to those of nonfatty cells in the same cultures. As the adipose conversion proceeds, the processes thicken and retract; the cells eventually acquire the rounded shape of the more mature adipose cells.     

Loss of choleragen receptors and ganglioside upon differentiation of 3T3-L1 preadipocytes

3T3-L1 preadipocytes differentiate in culture into cells having the enzymatic and morphological characteristics of adipocytes. Differentiation is accompanied by a decrease in total cellular ganglioside content; the ganglioside level is 1.8 to 2.5-fold higher in undifferentiated than in differentiated cells. Gangliosides GM3 and GD1a constitute a majority of total cell gangliosides in both cell types, while ganglioside GM1, the putative choleragen receptor, constitutes less than 5%. Differentiation results in a 75 to 85% decrease in ganglioside GM1. An inverse correlation exists between the percentage of adipocytes in the cell population and: 1) total ganglioside and ganglioside GM1 content, and 2) surface ganglioside GM1 as estimated by choleragen binding or fluorescent staining of bound choleragen. Nondifferentiating 3T3-C2 control cells do not exhibit changes in total ganglioside, ganglioside GM1, or choleragen binding that are observed with 3T3-L1 cells.