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Endocrinology Vol. 144, No. 6 2559-2565
Copyright © 2003 by The Endocrine Society

Mechanism of Adult Primitive Mesenchymal ST-13 Preadipocyte Differentiation

Yukiko Yajima, Mayumi Sato, Michihiro Sumida and Seiichi Kawashima

Department of Molecular Biology (Y.Y., S.K.) and Pharmaceutical Research and Development Center (M.S.), Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan; and Second Department of Medical Biochemistry, Ehime University School of Medicine (M.S.), Shigenobu-cho, Onsen-gun, Ehime 791-0295, Japan

Address all correspondence and requests for reprints to: Dr. Yukiko Yajima, Tokyo Metropolitan Institute of Medical Science, 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113-8613, Japan. E-mail: yajima{at}rinshoken.or.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Convincing evidence supports the idea that adipogenesis occurs throughout the life of organisms. However, little is known about the adipogenesis program for adult adipocytes. We examine this issue using mouse adult primitive mesenchymal ST-13 preadipocytes that express the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) gene while in a predifferentiated state. The gene expression of PPAR{gamma} was sustained throughout differentiation when ST-13 preadipocytes were induced to become adipocytes by a PPAR{gamma} ligand. However, the differentiation of pluripotent C3H10T1/2 stem cells and 3T3-L1 embryonic fibroblastic cells was associated with enhanced expression of the PPAR{gamma} gene. Immunoblotting analysis revealed that C3H10T1/2 and 3T3-L1 cells expressed low levels of PPAR{gamma}1 from the early stage, and the amount increased during differentiation, whereas PPAR{gamma}2 appeared at the late stage. In contrast, ST-13 preadipocytes expressed an appreciable amount of PPAR{gamma}1 that significantly decreased on differentiation, and a small amount of PPAR{gamma}2 appeared late in the differentiation process. Furthermore, the standard hormone cocktail containing dexamethasone, methylisobutylxanthine, and insulin induced an increase in PPAR{gamma}1 protein only at the early stage, and a low level of PPAR{gamma}2 protein appeared late in ST-13 cells. However, levels of both PPAR{gamma}1 and PPAR{gamma}2 proteins were significantly induced within 2 d in 3T3-L1 cells in this hormonal adipogenesis. Moreover, exposing ST-13 preadipocytes to dexamethasone and insulin induced differentiation, but failed to induce adipogenesis in 3T3-L1. Adipogenesis in adult rat primary preadipocytes was also induced in a similar manner to that of ST-13. Our results indicate that ST-13 cells and primary preadipocytes derived from adults possess an adipogenesis program distinct from that of 3T3-L1 and C3H10T1/2 cells, and that it may represent the adipogenesis program for adult-specific adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING ADIPOCYTE differentiation, a large number of genes have to be regulated in a selective and coordinated manner, and cell morphology as well as gene expression undergo significant changes (1). Regulatory regions of adipose-specific genes have been characterized. This has led to identification of the transcription factors, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and CCAAT/ enhancer binding protein (C/EBP), which play key roles in the complex transcriptional cascade of adipocyte differentiation (2, 3, 4). The 3T3-L1 preadipocyte cell line was clonally isolated from Swiss-3T3 cells derived from disaggregated 17- to 19-d-old mouse embryos (5, 6). This line has been used the most frequently as a model for adipogenesis research. During the differentiation of adipocytes from 3T3-L1 cells determined using a standard protocol including dexamethasone (DEX), 1-methyl-3-isobutylxanthine (MIX), and insulin, three C/EBP isoforms and PPAR{gamma} are sequentially expressed; C/EBPß and -{delta} are induced temporarily at an early stage, and this is followed by PPAR{gamma} (7, 8). C/EBP{alpha} is expressed at the late stage of differentiation (7). The role of PPAR{gamma} in the initiation of differentiation has been documented (9). Two isoforms ({gamma}1 and {gamma}2) of this protein have been translated from at least three mRNAs, which are generated by alternative splicing of the same gene product. PPAR{gamma}1 and -{gamma}2 have almost identical amino acid sequences, but the N terminus of {gamma}2 has an additional 30 amino acids (10). The transcriptional product of the PPAR{gamma} gene has been detected in many tissues, and {gamma}1 is the predominant isoform. PPAR{gamma}2 is highly fat-selective and is expressed at very high levels in fat tissue (11). A member of the nuclear receptor family, PPAR{gamma} is activated through ligands that bind to its distinct domain at the carboxyl terminal. These ligands include natural prostaglandin J2 derivatives (12, 13), some nonsteroidal antiinflammatory drugs (14), and antidiabetic thiazolidinediones (15, 16). All of these have similar PPAR{gamma}1 and -{gamma}2 binding potencies (17). Both isoforms of PPAR{gamma} form an obligate heterodimer with the retinoid X receptor to bind to regulatory elements within the promoters/enhancers of many genes associated with lipid metabolism (18). The ability of PPAR{gamma} for transcriptional activity is located in the NH2-terminal region, and is ligand independent. PPAR{gamma}2 is about 10-fold more active than PPAR{gamma}1 in terms of ligand-independent transcriptional activation, and this effect requires insulin (19). In addition, a key role has been suggested for PPAR{gamma}2 in driving the initiation of adipogenesis (20, 21).

Adipogenesis occurs in both the prenatal and postnatal states and also occurs throughout the life span of organisms (1). The homozygous null mutation of PPAR{gamma} is lethal on embryonic d (E) 9.5–E10, before the stage where the establishment of embryonic fibroblasts is generally considered feasible to adipogenesis (22, 23). Therefore, adipogenesis of embryonic fibroblasts may be essential for life. On the other hand, the modulation of fat cell differentiation of adult preadipocytes can have profound effects at extraadipose sites. One such example found in a transgenic mouse is the association of lipoatrophy with systemic insulin resistance and diabetes (24). However, the precise adipogenesis program for primitive adult preadipocyte cells remains unknown. The present study used mouse primitive mesenchymal ST-13 preadipocytes established from adult murine mammary cells (25). These preadipocytes can undergo differentiation to adipocytes in the presence of the PPAR{gamma} ligand, ciglitadione (15). However, this cell line has not yet been characterized in terms of transcription factors. We compared the process of ST-13 cell differentiation with that of pluripotent C3H10T1/2 stem cells and 3T3-L1 embryonic fibroblast cells. Our results demonstrated that although the three types of preadipocytes have a morphologically defined adipocyte phenotype, only ST-13 cells express PPAR{gamma}1 as preadipocytes, and PPAR{gamma}1 might be responsible for the initiation of adipogenesis in ST-13 cells. Rat primary preadipocytes were also induced to undergo adipogenesis in a manner similar to that of ST-13 differentiation. Our results indicate that the adipocyte pathway of adult-derived ST-13 cells represents the adipogenesis program for adult-specific adipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Drugs and chemicals were obtained from the following sources: insulin, DEX, MIX, fetal bovine serum, calf serum, culture media, and BSA (fraction V, essentially fatty acid free) from Sigma-Aldrich Corp. (St. Louis, MO); PPAR{gamma} antibody (catalog no. sc-7273) and C/EBP{alpha} antibody (catalog no. sc-61) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and alkaline phosphatase-conjugated secondary antibodies and Vector substrate kit from Promega Corp. (La Jolla, CA). The thiazolidinedione derivative, ciglitazone (5-[4-(t-3-hydroxyl-methyl-r-1- cyclohexyl-methoxy)benzyl]-2,4-thiazolidinedione) was a gift from Takeda Chemical Industries, Ltd. (Osaka, Japan).

Cell culture
Pluripotent C3H10T1/2 stem cells and 3T3-L1 embryonic fibroblast cells were obtained from the American Type Culture Collection (Manassas, VA). ST-13 was established from adult primitive mesenchymal cell lines and has been used in vitro to study adipocytic conversion (25, 26). These three cell lines were maintained under an atmosphere of 5% CO2 in DMEM-Ham’s F-12 nutrient medium (1:1 mixture) containing 25 mM glucose and supplemented with 10% calf serum. To induce adipocyte differentiation, all three murine preadipocytes were treated at confluence for 7–10 d with ciglitazone (1 µg/ml) and insulin (4 µg/ml) in DMEM-Ham’s F-12 containing 10% fetal bovine serum. Medium was replenished with ciglitazone and hormone every 2 d. ST-13 and 3T3-L1 preadipocytes were also cultured and induced to differentiate with a standard hormone cocktail containing 1 µM DEX, 0.5 mM MIX, and 1 µg/ml insulin or combinations of these inducers for 2 d. After incubation with these reagents, the basal medium containing 10% fetal bovine serum was replenished every other day. Differentiated adipocytes were stained with Oil Red O (27). Primary preadipocytes isolated from the sc adipose tissue of Wistar male rats (8 wk old) were supplied by Toyobo Co. (Tokyo, Japan) and cultured in synthetic growth medium as described in the supplier’s manual. For adipocyte differentiation experiments, cells were seeded into cell culture dishes at a density of 3.5 x 104 cells/cm2 with growth medium. At confluence, the medium was changed to a synthetic basal medium supplemented with 1% FBS, the standard hormone cocktail (DEX, MIX, and insulin), or a combination of these inducers for 2 d. Thereafter, the synthetic medium supplemented with 1% FBS was replenished every other day. Triacylglycerol was quantified using triglyceride test kit (Wako Co., Tokyo, Japan).

Northern blot analysis
Total RNA was extracted using the method of Chomczynski and Sacchi (28). RNA was separated on a 1% formaldehyde agarose gel and then transferred to a Biodyne B membrane (Pall BioSupport Division, Port Washington, NY). cDNA probes were labeled with random 9-mers (Takara Co., Tokyo, Japan) and [{alpha}-32P]deoxy-CTP. Hybridization proceeded at 65 C in rapid hybridization buffer (Amersham International, Little Chalfont, UK) according to the manufacturer’s instructions. The membrane was washed with 2x standard saline citrate and 0.1% SDS at room temperature, followed by 0.1x standard saline citrate and 0.1% SDS at 55 C. An image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan) detected radioactive bands. A cDNA probe for mPPAR{gamma}2-(40–879) was prepared from genomic DNA. The cDNAs for C/EBP{alpha}, C/EBPß, and adipocyte protein 2 (aP2) were supplied by Dr. Masahiko Kuroda (Tokyo University, Tokyo, Japan) (29) and that for C/EBP{delta} was supplied by Dr. Naohiro Yamamoto (Osaka University, Osaka, Japan).

Western blot analysis
Nuclear extracts were prepared as described by Engelman et al. (30). Briefly, cells in two 60-mm confluent dishes were harvested and resuspended in 0.3 ml cold buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 µM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 2.5 µg/ml aprotinin]. Thereafter, 20 µl 10% Nonidet P-40 solution were added, and the cell suspension was rocked vigorously for 10 sec. Nuclei were recovered by centrifugation for 30 sec at 4 C and subsequently resuspended in 150 µl ice-cold buffer C [20 mM HEPES (pH 7.9), 0.4 M NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1 µM dithiothreitol plus protease inhibitors, as described above] and rocked vigorously at 4 C for 30 min. The nuclear lysate was centrifuged at 100,000 rpm for 12 min in a TL-100 ultracentrifuge (Beckman, Palo Alto, CA) at 4 C, and the supernatant was divided into aliquots and stored at -80 C. Nuclear extracts were separated on SDS-polyacrylamide gels (10%) and transferred to Immobilon-P transfer membranes. Nonspecific binding on the membrane was blocked with 5% skim milk in Tris-buffered saline for 1 h at room temperature. After incubation with the primary antibody, anti-PPAR{gamma} or anti-C/EBP{alpha} antibody, binding was detected using alkaline phosphatase-conjugated secondary antibodies and the Vector substrate kit. Preliminary experiments established that the intensities of these transcriptional factors were directly proportional to the amount of nuclear protein (from 3–20 µg nuclear proteins for C/EBP{alpha} and PPAR{gamma}1 and -{gamma}2 in 3T3-L1, C3H10T1/2, and ST-13 adipocytes, and from 7.7–30.8 µg nuclear proteins for C/EBP and PPAR{gamma}1 and {gamma}2 in rat primary adipocytes).

Protein determination
Protein was determined by the Bradford dye method (Bio-Rad Laboratories, Inc., Richmond, CA) using BSA as the standard.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PPAR{gamma} ligand-induced adipocyte conversion and expression of adipogenic genes
We induced terminal adipocyte differentiation using ciglitazone. Confluent adult primitive mesenchymal ST-13 preadipocytes were cultured in medium containing 1 µg/ml PPAR{gamma} ligand, ciglitazone, 4 µg/ml insulin, and 10% fetal bovine serum as adipogenic inducers. Conversion was monitored using Oil Red-O staining. Figure 1Go shows that most cells exhibited the adipocyte phenotype (cell rounding and accumulation of cytoplasmic lipid droplets) when cultured with the adipogenic inducers. The adipose conversion of pluripotent C3H10T1/2 stem cells and 3T3-L1 embryonic fibroblastic cells was similar to that of ST-13 cells accompanying the accumulation of triacylglycerol of 10.3 ± 0.5 mg/DNA mg in ST-13, 6.5 ± 0.3 mg/DNA mg in 3T3-L1, and 10.8 ± 1.5 mg/DNA mg in C3H10T1/2, respectively, after 9-d treatment of these inducers (Fig. 1Go). ST-13 and C3H10T1/2 adipocytes accumulated more lipid than 3T3-L1 cells. On the other hand, the differentiation of ST-13 cells as well as that of C3H10T1/2 and 3T3-L1 cells were inhibited by TNF{alpha} (data not shown). We then examined the temporal expression of the adipogenic transcription factors and adipocyte-specific gene, aP2, during the differentiation of ST-13, C3H10T1/2, and 3T3-L1 preadipocytes. Representative Northern blots are shown in Fig. 2Go. Levels of C/EBP{delta} mRNA, which were high in the three preadipocyte cell lines at confluence (d 0), were modestly diminished at differentiation. C/EBPß gene expression in 3T3-L1 cells transiently peaked on d 1 and decreased to 30–50% of the original prestimulatory levels between d 4 and 6. The pattern of C/EBPß expression was similar during ST-13 cell differentiation. In contrast, C3H10T1/2 cells expressed C/EBPß mRNA in a different manner, starting to increase on d 3 after exposure to the adipogenic inducers and reaching a maximum level between d 4–8. The C/EBP{alpha} gene was similarly induced on d 3 and continued to increase to the maximum level between d 4 and 8 in all three cell lines. Expression of the PPAR{gamma} gene in ST-13 cells was quite different from that in C3H10T1/2 and 3T3-L1 cells. PPAR{gamma} expression in C3H10T1/2 and 3T3-L1 cells increased around d 1–3 and reached the maximum level on d 4. However, ST-13 cells expressed the PPAR{gamma} gene during the predifferentiated state. The level decreased between d 3 and 4 after exposure to the adipogenic inducers and then weakly increased between d 6–8. The expression of the gene for adipocyte-specific fatty acid-binding protein, aP2, was induced early in the differentiation process of all preadipocyte cell lines, confirming adipocyte differentiation.



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  		Figure 1. The thiazolidinedione derivative, ciglitazone, promotes adipocyte differentiation in three preadipocyte cell lines. Pluripotent C3H10T1/2 stem cells, 3T3-L1 embryonic fibroblasts, and ST-13 adult primitive mesenchymal cells were incubated for 9 d with vehicle alone (0.1% dimethylsulfoxide) or 1 µg/ml ciglitazone and 4 µg/ml insulin (see Materials and Methods), then lipid droplet accumulation was identified by staining with Oil Red-O.

 


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  		Figure 2. Induction of adipogenic transcription factors and adipocyte gene expression during differentiation. The cell lines described in Fig. 1Go were cultured after reaching confluence in differentiation medium for 8 d. Total RNA (10 µg/lane) from C3H10T1/2, 3T3-L1, and ST-13 cell lines was isolated at the indicated times, resolved by electrophoresis, blotted to nylon membranes, and hybridized with {alpha}-32P-labeled cDNA probes. Equivalent amounts of RNA (10 µg/lane) were loaded as indicated during ethidium bromide (EtBr) staining of ribosomal RNAs (bottom). The figure shows representative data from three separate experiments.

 
Immunoblotting analyses of PPAR{gamma}1 and PPAR{gamma}2 proteins during adipocyte differentiation
Because the probe for PPAR{gamma} used in the Northern blotting could not distinguish PPAR{gamma}1 and PPAR{gamma}2 mRNAs, we examined the PPAR{gamma} proteins by immunoblotting (Fig. 3AGo). A trace amount of PPAR{gamma}1 protein was detected in preadipocytes of C3H10T1/2 and 3T3-L1 cells. Expression of the PPAR{gamma}1 protein increased within 2 d of the differentiation course and reached a maximum at 4–6 d, whereas PPAR{gamma}2 protein appeared later in the differentiation process (d 6) in both cell lines (Fig. 3BGo). ST-13 cells already expressed a high level of PPAR{gamma}1 protein at confluence (d 0), and ciglitazone caused a sustained fall in PPAR{gamma}1 that decreased by 62.0 ± 6.7% on d 6. ST-13 cells also contained a low level of PPAR{gamma}2 protein at 6–8 d. These data are consistent with those from the Northern blots of PPAR{gamma} mRNA (Fig. 2Go). Ciglitazone on ST-13 cells also similarly affected the other PPAR{gamma} ligands, BRL-49653 and prostaglandin J2 (data not shown). The expression of C/EBP{alpha} protein was induced during differentiation with a time course similar to those of all preadipocyte cell lines (Fig. 3BGo).



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  		Figure 3. Expression of PPAR{gamma} and C/EBP{alpha} proteins during adipocyte conversion of C3H10T1/2, 3T3-L1, and ST-13 cells. The three cell lines described in Fig. 1Go were cultured in the differentiation medium for 8 d postconfluence. Nuclear extracts from C3H10T1/2, 3T3-L1, and ST-13 cell lines were isolated at the indicated times. A, Equal amounts of nuclear extract (10 µg protein) were resolved by gel electrophoresis and immunoblotted using anti-PPAR{gamma} and anti-C/EBP{alpha} antibodies. B, Quantitation of PPAR{gamma}1 and PPAR{gamma}2 (upper), and C/EBP{alpha} (lower), by densitometric analysis from three independent experiments. PPAR{gamma}1, PPAR{gamma}2, and C/EBP{alpha} protein levels are expressed as arbitrary units, and data points are the mean ± SEM of three separate experiments.

 
Effects of hormonal stimulants on the process of adipocyte conversion and PPAR{gamma} expression
We exposed confluent ST-13 and 3T3-L1 preadipocytes to a standard hormone cocktail containing DEX, MIX, and insulin, or combinations of these inducers to determine differences in the mechanisms of adipogenesis. The cells were maintained according as described in Materials and Methods, and the nuclear fraction was extracted at the indicated times after induction. When treated with the standard hormonal cocktail, both cell lines differentiated to an adipocyte phenotype, but the omission of DEX or MIX from the standard mixture significantly attenuated the differentiation of 3T3-L1 preadipocytes into mature fat cells, as indicated by their phenotype (Fig. 4AGo). Percentages of lipid-containing cells were 5.0 ± 1.7% for MIX and insulin treatment, 15.6 ± 2.8% for DEX and insulin, and 89.0 ± 13.1% for MIX, DEX, and insulin. In the case of ST-13 cells, omitting MIX from the standard mixture resulted in a degree of lipid accumulation similar to that achieved when exposed to the standard hormone mixture (Fig. 4AGo). However, evidence of differentiation was minimal in ST-13 preadipocytes cultured without DEX (Fig. 4AGo). Percentages of lipid-containing cells were 27.4 ± 5.4% for MIX and insulin treatment, 70.4 ± 10.5% for DEX and insulin, and 85.0 ± 14.1% for MIX, DEX, and insulin. During the adipogenesis of 3T3-L1 cells exposed to the standard hormonal cocktail, the expression of PPAR{gamma}1 and PPAR{gamma}2 proteins increased dramatically within 2 d of differentiation, and continued to increase during adipocyte differentiation (Fig. 4Go, B and C). In contrast, ST-13 cells exhibited a high level of PPAR{gamma}1 protein in preadipocytes; this level was amplified further, with an increase of about double on d 4 of differentiation in the standard hormonal cocktail. The induction of PPAR{gamma}2 protein was later (d 4), and it reached 38 ± 2.5% of the PPAR{gamma}1 levels (d 7). Of particular interest, the early expression of PPAR{gamma}1 protein in ST-13 cells treated with DEX and insulin increased, whereas PPAR{gamma}2 protein was expressed late and at a very low level. The ST-13 preadipocytes seemed to be converted to adipocytes in a PPAR{gamma}1-dependent manner.



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  		Figure 4. Effects of hormonal inducers on adipocyte conversion and PPAR{gamma} expression. ST-13 and 3T3-L1 cells were induced to differentiate in medium containing insulin (I) and FBS in the presence or absence of DEX (D) and MIX (M). Eight days later, cells were stained with Oil Red-O (A). Nuclear fractions were prepared from ST-13 and 3T3-L1 cells during differentiation at the indicated time points. Equal amounts of nuclear extract (10 µg protein) were resolved by gel electrophoresis and immunoblotted using anti-PPAR{gamma} and anti-C/EBP{alpha} antibodies (B). Protein levels of PPAR{gamma}1, PPAR{gamma}2, and C/EBP{alpha} were quantified by densitometric analysis and expressed as arbitrary units. Each column is the mean ± SEM of three separate experiments (C).

 
Effects of hormonal stimulants on the adipocyte conversion of rat primary preadipocytes
To determine whether the adipogenesis program in ST-13 cells is universal for adult adipocytes, we incubated primary preadipocytes isolated from the sc adipose tissue of the adult rat with a combination of hormones. Figure 5AGo shows that the rat primary preadipocytes converted to adipocytes in the presence of the standard hormonal cocktail or DEX combined with insulin, but these primary preadipocytes could not undergo adipogenesis in the absence of DEX (percentages of lipid-containing cells, 3.7 ± 0.9% for MIX and insulin treatment, 54.0 ± 13.0% for DEX and insulin, and 33.3 ± 12.6% for MIX, DEX, and insulin). The PPAR{gamma}1 protein that was detected in this primary preadipocytes, but at a low level unlike that in ST-13 preadipocytes, increased within 3 d of differentiation and continued to increase whenever cells were exposed to the standard hormonal cocktail or DEX plus insulin (Fig. 5Go, B and C). On the other hand, a very low level of PPAR{gamma}2 was expressed during both adipogenic processes. Amounts of C/EBP{alpha} proteins also increased in the rat primary preadipocytes during adipogenesis (Fig. 5Go, B and C). This was also similar to the profile of adipogenesis in primary preadipocytes from perirenal adipose tissue (data not shown). These results indicated that the primary preadipocytes isolated from adult rat adipose tissue were induced to adipocytes in a similar manner as adipogenesis of ST-13.



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  		Figure 5. Effects of hormonal inducers on conversion of rat primary preadipocytes into adipocytes. Rat primary preadipocytes were induced to differentiate in medium containing insulin (I) and FBS in the presence or absence of DEX (D) and MIX (M). Eight days later, cells were stained with Oil Red-O (A). Nuclear extracts from rat primary preadipocytes were prepared during differentiation at the indicated times. Equal amounts of nuclear extract (10 µg protein) were resolved by gel electrophoresis and immunoblotted using anti-PPAR{gamma} and anti-C/EBP{alpha} antibodies (B). Protein levels of PPAR{gamma}1, PPAR{gamma}2, and C/EBP{alpha} were quantified by densitometric analysis and expressed as arbitrary units. Each column is the mean ± SEM of three separate experiments (C).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been reported that embryonic fibroblasts isolated from 12- to 13-d-old embryos have the capacity to differentiate into adipocytes despite the absence of a clear molecular marker for preadipocytes (1). On the other hand, primary adult preadipocytes were isolated from various species at different stage of maturation. These cells are similar in many regards, but their responsiveness to inducing agents varies considerably (31). Primary preadipocytes from human adipose tissue need only a few hormonal signals for differentiation without requiring additional replication steps for the initiation of differentiation (32). It seems that the majority of adult preadipocytes isolated from fat pads has already entered terminal differentiation in vivo and may be in a late stage of the adipocyte program while embryonic fibroblasts are at an early stage. Therefore, the adipogenesis programs should be different between adult and embryonic primary preadipocyte cells. In this study, we discovered that adult-derived ST-13 cells possess an adipogenesis program distinct from that of 3T3-L1 and C3H10T1/2 cells, and this may represent the precise adipogenesis program for adult adipocytes. We showed that a PPAR{gamma} ligand, ciglitazone, induced adipogenesis in three preadipocyte cell lines. In C3H10T1/2 and 3T3-L1 cells, ciglitazone promoted an extreme increase in PPAR{gamma}1 protein during adipogenesis, and PPAR{gamma}2 appeared only late in the process (d 6–8). The induction of C/EBP{alpha} seemed to correspond with that of PPAR{gamma}1, not of PPAR{gamma}2 (Fig. 3BGo). The ST-13 adult primitive mesenchymal cell line, however, has a completely different pattern of PPAR{gamma} mRNA and protein expression. ST-13 cells express an appreciable level of PPAR{gamma}1 protein at the preadipocyte stage, and an adipogenic inducer, ciglitazone, induced a moderate decrease in PPAR{gamma}1 expression and a slight appearance of PPAR{gamma}2 at the late stage of differentiation (d 6). Camp et al. (33) speculated that during the course of differentiation there is a threshold point for which a specific level of PPAR{gamma} activity is required for adipogenesis to proceed, and PPAR{gamma} activators enhance the rate at which this threshold is overcome. Upon attainment of the required level of PPAR{gamma} activity, the continued presence of ligands causes receptor down-regulation similar to that seen for other nuclear receptors (33). Therefore, it is likely that ciglitazone may activate the preexisting PPAR{gamma}1 early in ST-13 preadipocytes, and this triggers the initiation of adipogenesis. The down-regulation of PPAR{gamma}1 in ST-13 cells is consistent with the observation that troglitazone treatment caused a significant decrease in PPAR{gamma} proteins and DNA-binding activity (33, 34). However, it seems that C3H10T1/2 and 3T3-L1 cells do not have sufficient levels of PPAR{gamma}1 to be down-regulated during adipocyte differentiation.

Next, we again confirmed the specific pathway for ST-13 adipocyte differentiation. When the hormonal adipogenic inducers stimulated ST-13 as well as 3T3-L1 preadipocytes, cells produced increased levels of PPAR{gamma}1 protein and rarely expressed PPAR{gamma}2 at the early stage. On the other hand, considerable amounts of PPAR{gamma}1 and PPAR{gamma}2 proteins appeared in 3T3-L1 cells within 2 d in the presence of standard hormonal adipogenic inducers (Fig. 4Go, B and C). This correlates well with published reports describing the early induction of PPAR{gamma}2 mRNA and protein during 3T3-L1 cell differentiation (20, 35). Furthermore, we demonstrated that omitting MIX from the standard cocktails in which ST-13 preadipocytes or primary adult preadipocytes were incubated induced large amounts of PPAR{gamma}1, C/EBP{alpha}, and a corresponding conversion of the preadipocytes into morphologically distinct adipocytes. However, 3T3-L1 cells did not convert to adipocytes under the same conditions. Hamm et al. (36) demonstrated that exposing 3T3-L1 preadipocytes to DEX and insulin does not induce adipogenesis. However, incubating MIX-deficient 3T3-L1 preadipocytes with PPAR{gamma} ligand rescues the block of adipogenesis (36). Therefore, ST-13 adult primitive or adult primary preadipocytes appear to have acquired the ability to produce an appropriate ligand for PPAR{gamma}, whereas 3T3-L1 cells cannot produce an endogenous ligand(s) or activators. Our results showed that ST-13 and 3T3-L1 cells have distinct adipogenesis programs. The activation of PPAR{gamma}1 in ST-13 preadipocytes seems to be involved in the initiation of a transcriptional cascade that results in the induction of PPAR{gamma}2 and C/EBP{alpha} expression, when PPAR{gamma} ligands or hormonal inducers are the source of stimulation. On the other hand, 3T3-L1 adipocyte differentiation might proceed through at least two pathways: one would be regulated by PPAR{gamma} ligands that activate PPAR{gamma}1 to accelerate adipogenesis, and the other is via hormonal inducers that may involve the activation of genes such as PPAR{gamma}2 rather than PPAR{gamma}1 (20, 33). These observations are in good agreement with the suggestion by Saladin et al. (20) that PPAR{gamma}1 and PPAR{gamma}2 have different functions, with PPAR{gamma}1 being used when ligand is abundant, and PPAR{gamma}2 being important under conditions of low ligand concentrations. It is conceivable that PPAR{gamma}1 is the most abundant isoform in preadipocytes in vivo, because its mRNA represents more than 80% of the PPAR{gamma} transcripts in sc adipose tissue and isolated adipocytes (37). This study showed that PPAR{gamma}1 might be responsible for the initiation of adipogenesis in the adult primitive mesenchymal preadipocyte cell line, ST-13, or in primary preadipocytes from adult rat adipose tissue.


   Acknowledgments
 
We thank Drs. T. Ono and N. Suzuki for valuable advice regarding Northern blot protocols, and Mr. S. Isomura for excellent technical assistance.


   Footnotes
 
Abbreviations: aP2, Adipocyte protein 2; C/EBP, CCAAT/enhancer binding protein; DEX, dexamethasone; E, embryonic day; MIX, 1-methyl-3-isobutylxanthine; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}.

Received August 26, 2002.

Accepted for publication February 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rosen ED, Spiegelman BM 2000 Molecular regulation of adipogenesis. Annu Rev Cell Dev Biol 16:145–171[CrossRef][Medline]
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