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Distinct Transcriptional Profiles of Adipogenesisin Vivo and in Vitro

Distinct Transcriptional Profiles of Adipogenesisin Vivo and in Vitro THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 36, Issue of September 7, pp. 34167–34174, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Distinct Transcriptional Profiles of Adipogenesis in Vivo and in Vitro* Received for publication, May 15, 2001 Published, JBC Papers in Press, July 9, 2001, DOI 10.1074/jbc.M104421200 Alexander Soukas‡, Nicholas D. Socci§¶, Barbara D. Saatkamp‡, Silvia Novelli‡, and Jeffrey M. Friedman‡** From the ‡Laboratory of Molecular Genetics, the §Center for Studies in Physics and Biology, and the Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, and the ¶Institute of Theoretical Physics, University of California, Santa Barbara, California 93106 Obesity, defined as an increase in adipose tissue mass, esis is the result of a temporally ordered pattern of 3–5 distinct is the most prevalent nutritional disorder in industrial- phases of gene expression (summarized in the above reviews). ized countries and is a growing problem in developing Indeed several of the more than 100 molecules that have been countries. An increase in adipose tissue mass can be the identified as differentially expressed during the transition of result of the production of new fat cells through the preadipocytes to adipocytes in culture have been shown to have process of adipogenesis and/or the deposition of in- binding sites for the transcription factors PPAR and C/EBP creased amounts of cytoplasmic triglyceride per cell. in their promoters (9, 10). Although much has been learned about the differentia- Although cell culture models of adipocytes faithfully express tion of adipocytes in vitro, less is known about the mo- many genes that are markers of adipocytes in vivo, the events lecular basis for the mechanisms regulating adipogene- that trigger this transformation in vivo are not as well under- sis in vivo. Here oligonucleotide microarrays have been stood. Knockout studies of C/EBP, C/EBP, C/EBP, and used to compare the patterns of gene expression in prea- PPAR have confirmed that these molecules are necessary in dipocytes and adipocytes in vitro and in vivo. These data vivo for adipogenesis, but it is not known with certainty indicate that the cellular programs associated with adi- whether these factors are sufficient (11–15). Thus it is unclear pocyte differentiation are considerably more complex whether the high level of expression of these factors evident in than previously appreciated and that a greater number in vitro adipocytes can recapitulate the gene expression profile of heretofore uncharacterized gene regulatory events of adipocytes in vivo. It has already been shown that some are activated during this process in vitro. In addition, adipocyte-derived molecules such as leptin are expressed at the gene expression changes associated with adipocyte lower levels in cultured adipocytes (16) and that in vivo levels development in vivo and in vitro, while overlapping, are in some respects quite different. These data further sug- of ob mRNA are restored in fat pads derived from subcutane- gest that one or more transcriptional programs are ac- ously implanted 3T3-F442A preadipocytes (17). tivated exclusively in vivo to generate the full adipocyte To characterize the regulation of gene expression during phenotype. This gene expression survey now sets the adipogenesis in vivo and in vitro further, the abundance of stage for further studies to dissect the molecular differ- 11,000 genes and expressed sequence tags was measured at 10 ences between in vivo and in vitro adipocytes. different time points during in vitro 3T3-L1 adipocyte differen- tiation using oligonucleotide microarrays (Affymetrix, Santa Clara, CA). The abundance of the same 11,000 genes was also Adipogenesis has been studied extensively in vitro using a measured in adipocytes and stromal cells (including preadipo- number of preadipocyte cell lines including 3T3-L1 cells (1). cytes) isolated from wild-type and ob/ob white adipose tissue. When cultured in defined media, 3T3-L1 cells deposit triglyc- Independent analyses of these data indicated that a more com- eride in cytoplasmic lipid droplets and express genes that are plex program of gene expression than was known previously is also expressed in adipocytes in vivo (2– 8). Elegant studies of activated during adipocyte differentiation in vitro and in vivo. this process have led to the identification of several key regu- Comparative analysis of the in vitro and in vivo expression latory genes that are necessary and/or sufficient for the tran- data revealed that although some genes are expressed at in sition of preadipocytes into adipocytes in vitro including vivo levels in fully differentiated 3T3-L1 cells, most notably CCAAT/enhancer binding proteins (C/EBPs) , , and  and target genes for C/EBP and PPAR , large clusters of genes peroxisome proliferator-activated receptor (PPAR)  (9). Stud- are expressed at much higher levels or even exclusively in vivo. ies of these transcription factors have suggested that adipogen- Conversely, a large group of genes is expressed in vitro that is not or is poorly expressed in vivo in adipocytes or preadipo- cytes. Although these data represent a descriptive survey of * This work was supported by NINDS National Institutes of Health Grant NS39662 (to J. M. F and N. D. S), MSTP National Institutes of gene expression profiles of adipogenesis in vivo and in vitro, Health Grant GM07739 (to A. S.) and by NSF National Institutes of together they indicate that adipogenesis is likely to be more Health Grant PHY99-07949 (to N. D. S). The costs of publication of this complex than previously appreciated and that specific tran- article were defrayed in part by the payment of page charges. This scriptional programs that generate the full adipocyte pheno- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. type are not activated in cultured adipocytes but are activated ** To whom correspondence should be addressed: The Rockefeller by signals present exclusively in vivo. University, 1230 York Ave., Box 305, New York, NY 10021. Tel.: 212- 327-8800; Fax: 212-327-7420; E-mail: [email protected]. EXPERIMENTAL PROCEDURES The abbreviations used are: C/EBP, CCAAT/enhancer binding pro- 3T3-L1 Cell Culture—3T3-L1 cells (ATCC, Manassas, VA) were tein; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding protein. maintained in subconfluent cultures in Dulbecco’s modified Eagle’s This paper is available on line at http://www.jbc.org 34167 This is an Open Access article under the CC BY license. 34168 Global Expression Profiles of Adipogenesis medium supplemented with 33 M biotin, 17 M calcium pantothenate, The initial analysis of the expression data from the oligonu- 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomy- cleotide arrays indicated that the abundance of 1259 genes cin, and 0.25 g/ml amphotericin B (Life Technologies, Inc.). For differ- changed 3-fold or more during the course of the differentiation entiation, 3  10 cells were seeded per 100-mm plate, allowed to reach response. These data corroborated many previously reported 100% confluence, and induced 1 day post confluence with the above patterns of gene expression including changes of PREF1, M insulin, 2 nM triiodothyronine, 250 medium supplemented with 170 n nM dexamethasone, and 500 M isobutylmethylxanthine for 2 days (Life AEBP1, C/EBP, C/EBP, C/EBP, aP2, adipsin, Acrp30/Adi- Technologies, Inc.). After induction, the cells were fed every 2 days with poQ/adiponectin, lipoprotein lipase, hormone-sensitive lipase, maintenance medium supplemented with 170 nM insulin and 2 nM T3. stearoyl-CoA desaturase 1, 2 type VI collagen RNAs, and At indicated time points (Fig. 1), the medium was drained, the cells others (see below). were lysed by the addition of Trizol reagent (Life Technologies, Inc.), These data were analyzed using a modified k-means cluster- and total RNA was isolated as per protocol. Northern blotting was ing algorithm with a dot product metric, which groups genes conducted as described previously (18). For histology, cells were grown and differentiated as described above on lab-tek slides (Nunc, Naper- based on the similarity of their patterns of gene expression ville, IL), fixed (2% formaldehyde/0.2% glutaraldehyde in PBS for 15 (20). Cluster analysis indicated that the 1259 differentially min.), rinsed in PBS, stained with oil-red O (0.7% in 60% isopropanol for expressed genes can be grouped most parsimoniously into 27 10 min), and counter-stained with hematoxylin (40% in water for 5 temporally distinct patterns, each containing between 16 and min). 118 genes (Fig. 2), suggesting that the regulation of adipogen- Adipose Tissue Fractionation—Adipose tissue was divided into stro- esis may be considerably more complex than previously appre- mal and adipocyte fractions as described previously (19). Briefly, freshly excised peri-uterine fat pads from 8-week-old female C57Bl/6J, ciated. These clusters include 1000 genes not identified pre- wild-type, or ob/ob mice were rinsed in PBS, minced, and digested for 45 viously as being differentially regulated during adipocyte min–1hat37 °C in Krebs-Ringer bicarbonate (pH 7.4) with 4% bovine differentiation and more than 100 known transcription factors serum albumin and 1.5 mg/ml type I collagenase (Worthington, Free- and signaling molecules. hold, NJ). The digested tissue was filtered through a 250-m nylon 15 distinct clusters of RNAs increased in abundance during mesh to remove undigested tissue and centrifuged at 500  g for 5 min. adipogenesis in vitro (Fig. 2, left). 12 clusters of genes de- The floating adipocyte fraction was removed, washed in buffer, and recentrifuged to isolate free adipocytes. The stromal-vascular pellet was creased during adipogenesis (Fig. 2, right). Because of space resuspended in erythrocyte lysis buffer (154 mM NH4Cl, 10 mM restrictions, only a subset of the genes identified by this anal- KHCO3, and 1 mM EDTA), filtered through a 28-m nylon mesh to ysis are shown. All of the data are available online (arrays. remove endothelial cells, and pelleted at 500  g for 5 min. Total RNA rockefeller.edu/obesity/adipocyte). from the adipocyte and stromal fractions was isolated with Trizol rea- gent from five different ob/ob and wild-type preparations from which equal amounts were pooled for microarray analysis. Adequate separa- Patterns of Gene Expression in Adipocyte Differentiation tion of adipocyte and stromal fractions was confirmed by Northern Invoke Multiple Additional Transcriptional Mechanisms blotting for the adipocyte markers aP2 and PPAR (data not shown). Affymetrix Oligonucleotide Microarray Analysis and k-means Clus- Induced Genes—Microarray analysis indicated that the tran- tering—Samples were prepared for murine 11k microarrays from 10 g scription factors SREBP-1 (Fig. 2, A and C), C/EBP (Fig. 2C), of total RNA as outlined in the Affymetrix technical bulletin and as and PPAR2 (Fig. 2E) were up-regulated dramatically, each described previously (20). Hybridization and analysis were carried out with different kinetics, over the course of adipocyte differenti- using Affymetrix hybridization, washing, scanning, and Genechip 3.3 ation. Many genes that are markers of the differentiated adi- analysis stations as described in the Affymetrix technical manual. For 3T3-L1 analysis clustering, genes were included if greater than 3-fold pocyte increased in parallel with these factors. These included and more than 500 average difference change units (abundance meas- many known gene targets of these factors including the urement) increased or decreased in one time point relative to precon- SREBP-1 and C/EBP target genes fatty acid synthase (Fig. fluent expression levels. These genes were clustered according to fold 2C), stearoyl-CoA desaturase-1 (Fig. 2C), stearoyl-CoA desatu- change value using a modified k-means clustering algorithm with a dot rase-2 (Fig. 2D), the PPAR and C/EBP target gene aP2 (Fig. product metric described previously (20). For comparative in vivo and in 2E), and the highly adipocyte-enriched genes glycerophosphate vitro adipocyte and preadipocyte cluster analysis, genes were filtered to include only those genes that were either 5-fold enriched in in vivo dehydrogenase (Fig. 2E), adipsin (Fig. 2D), and Acrp30/Adi- adipocytes or preadipocytes (wild-type or ob/ob)or in vitro 3T3-L1 cells poQ/adiponectin (21) (Fig. 2D). at any time point relative to liver, hypothalamus, skeletal muscle, or However, many genes that mark the differentiated adipocyte pancreas. Genes were also included that were more than 5-fold different were expressed with different kinetics than SREBP-1, C/EBP, between in vivo preadipocytes and adipocytes (wild-type and ob/ob). and PPAR. For example, phosphoenolpyruvate carboxyki- Raw expression levels (average difference) in in vivo and in vitro sam- nase, a glycerogenic enzyme that has been shown to have a ples were set to 100 if they were below this value (to eliminate con- founding effects of background variations), log-transformed, baseline PPAR binding site in its promoter (22), is first expressed in normalized, and clustered (according to absolute abundance/hybridiza- adipocytes at 7 days and continues to increase in abundance tion intensity) with the above modified k-means clustering algorithm to until 28 days (Fig. 2G). The 3-adrenergic receptor, cytochrome group genes with similar patterns of expression. c oxidase VIIIH, glucose-6-phosphate isomerase, phosphofruc- tokinase I, and insulin-like growth factor II demonstrate a RESULTS similar pattern of expression and are also in this cluster. These 3T3-L1 Adipocyte Differentiation Occurs Through Multiple, patterns differed from the expression profile of the aforemen- Overlapping, Coordinated Phases of Gene Expression tioned transcription factors. These observations suggest that RNA was prepared from preconfluent and confluent preadi- other regulatory factors also play a role in adipogenesis. pocytes and from cells 6, 12, 24, and 48 h and 3, 4, 7, and 28 Repressed Genes—12 clusters of genes including cell cycle days after the cells were induced to the differentiate (Fig. 1A). genes (Fig. 2, Q and R), cytoskeletal genes (Fig. 2, Q and T), A standard differentiation protocol was optimized to maximize splicing factors and protein turnover genes (Fig. 2U), and the percentage of 3T3-L1 cells accumulating cytoplasmic lipid markers of other cell types including myelocytes and lympho- (greater than 90% at 7–28 days). Histologic staining for cyto- cytes (Fig. 2S) were down-regulated during differentiation. plasmic lipid confirmed that the cells had fully differentiated These clusters decrease with varying kinetics beginning as (Fig. 1A). Northern blotting with probes specific for aP2 and early as confluence (Fig. 2, P, R, W, X, and AA), after the PPAR, markers of mature adipocytes, confirmed that these addition of growth factors (Fig. 2Q), or later in the process of genes were expressed at in vivo levels in the differentiated cells adipocyte maturation (Fig. 2, S, T, U, V, Y, and Z). Although (Fig. 1B). several clusters contained transcription factors known to be Global Expression Profiles of Adipogenesis 34169 FIG.1. Time-course, histologic, and Northern analysis of 3T3-L1 differen- tiation. A, 3T3-L1 cells were seeded into 100-mm dishes at 3  10 cells/plate, and RNA was harvested at 10 points (indicat- ed by arrows) before or after initiation of differentiation by the addition of growth factors. Each of these samples was used for oligonucleotide microarray analysis. Histologic staining for lipid using oil-red O indicated that cells had begun to accu- mulate lipid by 3 days and had large cen- tral lipid droplets by 7–14 days. B, North- ern blotting for aP2 and PPAR of 10 g of total RNA indicated that the 3T3-L1 cells differentiated as described previ- ously (left panels) and expressed adipo- cyte genes at quantitatively similar levels relative to in vivo adipocytes (right pan- els). WT, wild type. repressed during adipogenesis including COUP-TF1 (Fig. 2Q) even C/EBP (up 3.6-fold) and C/EBP (up 3.9-fold) (Fig. 3B), and AEBP1 (Fig. 2Y), little is known about the transcriptional which have been invoked previously as responsible for mediat- mechanisms responsible for regulating these clusters of genes. ing the early phases of adipogenesis (23). All of these factors Transcription Factors and Signaling Molecules Regulated are transiently up-regulated after the addition of growth fac- During Adipogenesis—More than 100 known additional tran- tors to stimulate adipose conversion of confluent adipocytes. scription factors, transcriptional coactivators, and signaling Thyroid hormone receptor c-erbA-2 increased 13.8-fold prior molecules were regulated during adipogenesis. These factors to the addition of growth factors and remained elevated were either stably induced or repressed during differentiation throughout differentiation. Together, these results suggest the (Fig. 3, A, C, and D) or transiently regulated (Fig. 3, B and E). possible involvement of a large cluster of regulatory molecules In addition, in all clusters where a known adipogenic transcrip- and signaling pathways during differentiation. tion factor was present, additional transcription factors were Gene expression analyses indicated that a large group of coexpressed. Coordinate with a 71-fold increase in C/EBP and DNA binding inhibitors (Id genes) and high mobility group a 20.6-fold increase in SREBP-1, the transcription factors X- proteins (HMG genes) are down-regulated with distinct kinetic box-binding protein (up 6.4-fold), estrogen receptor-related  profiles during differentiation (Fig. 3C) These gene regulatory (up 4.2-fold), and Ig/enhancer binding protein (up 5.2-fold) were changes are accompanied by the transient or sustained down- up-regulated (Figs. 3A and 2C). The 18.1-fold induction of regulation of fos/jun family members, homeobox, forkhead, and PPAR was associated with a 15.3-fold up-regulation of the other transcription factors (Fig. 3, D and E). The role these transcriptional corepressor RIP140 and a 14.6- and 3.3-fold complex patterns play in generation of the differentiated adi- up-regulation of the transcription factors STAT-1 and iron pocyte remains to be determined. response element binding protein, respectively (Figs. 3A and Gene Expression in Adipocytes and Preadipocytes in Vivo 2E). In addition to these factors, which show similar profiles to C/EBP, SREBP-1, and PPAR, other transcription factors The phenotype of preadipocytes and adipocytes in vitro and were identified, which show different transcriptional profiles in vivo was compared by scoring the abundance of the same including Mxi-1, Zic3, and the albumin D-box-binding protein. 11,000 genes in RNA from the adipocyte and stromal (preadi- The glucocorticoid-induced leucine zipper (up 42.7-fold), N10 pocyte) fractions of C57Bl/6 wild-type and ob/ob mice. A total of nuclear hormone receptor (up 11.8-fold), and Wnt-4 signaling 1435 genes represented on the array were at least 5-fold en- molecule (up 34.8-fold) were induced to a greater extent than riched in adipocytes or preadipocytes as compared with other 34170 Global Expression Profiles of Adipogenesis tissues including liver, brain, skeletal muscle, and exocrine pancreas (see “Experimental Procedures”). k-means clustering of the absolute expression levels of these genes in adipocytes and preadipocytes in vivo and in vitro identified 18 distinct groupings of genes with significant expression levels. These included six clusters of genes that were enriched in adipocytes both in vivo and in vitro, five clusters of genes that were enriched in preadipocytes in vivo and in vitro, four clusters of genes that were specifically expressed in vivo, and three clus- ters that were expressed specifically in 3T3-L1 cells (Fig. 4). Genes Expressed in Vivo and in Vitro—This analysis con- firmed that for many genes, differentiated 3T3-L1 cells accu- rately recapitulate the in vivo patterns of gene expression observed in the transition of preadipocytes to adipocytes (Fig. 4A). Thus, many genes that are not expressed (or expressed at a low level) in preadipocytes were highly expressed in mature 3T3-L1 adipocytes, wild-type adipocytes in vivo, and ob/ob adi- pocytes in vivo (Table I). These six clusters of adipocyte-en- riched genes varied principally in their absolute level of expres- sion and were further divided into distinct subgroups by the k-means algorithm. The most notable members of this group include C/EBP, PPAR2, SREBP-1 (nonspecific SREBP-1a/1c probe set), and RXR. Many other adipocyte-specific and adi- pocyte-enriched genes are present in these groups including genes necessary to synthesize fatty acids from acetyl-CoA, the GLUT-4 glucose transporter, aP2, ACRP30/AdipoQ/adiponec- tin, and the 3-adrenergic receptor (Table I). A second group of five clusters were highly expressed in preadipocytes in vitro and in vivo and decreased in abundance during adipocyte conversion (Fig. 4B). These clusters include the transcriptional repressor AEBP1, which is negatively reg- ulated during adipogenesis and in preadipocytes serves to neg- atively regulate the aP2 AE-1 enhancer (24). The transcription factors Prx2 homeobox, junB, nuclear LIM interactor, Kruppel- like factor, forkhead box F2, E2a, and C/EBP are among others highly expressed in preadipocytes (prior to the accumu- lation of cytoplasmic lipid) and down-regulated during differ- entiation (Table I). Genes Expressed at High Levels in Vivo and Absent or Lower Levels in Vitro—68 genes that were highly expressed in adipo- cytes in vivo were expressed at an average of 20-fold lower levels in differentiated 3T3-L1 adipocytes (Fig. 4C, Vivo1). This cluster of genes indicates that adipocytes in vitro do not express the fully differentiated in vivo phenotype. This group of in vivo enriched genes included some genes encoding metabolic en- zymes such as ATP-citrate lyase (10.4-fold lower in 3T3-L1 day-28 adipocytes than wild-type adipocytes), phosphoenol- pyruvate carboxykinase (200-fold lower on day 7 and 1.9-fold lower on day 28), acetyl-CoA synthetase (3.1-fold lower on day 28), and leptin mRNA (63.4-fold lower on day 28). The high molecular weight growth hormone receptor and the thyrotropin receptor are 5.5- and 6.1-fold more highly expressed in vivo. Finally, the transcription factor skeletal muscle LIM protein FHL1 is 18.4-fold more highly expressed in wild-type adipo- cytes relative to 3T3-L1 day-28 adipocytes, which show only background hybridization levels. These results indicate that FIG.2. k-means cluster analysis of genes changing in abun- the absolute levels of expression of these genes are lower in dance during 3T3-L1 differentiation into adipocytes. Left, 1259 adipocytes in vitro, and although some are induced during genes were grouped into 27 clusters according to their profile of expres- sion across 10 time points by a modified k-means cluster algorithm. differentiation, additional signals seem necessary to direct Experiments are ordered along the x axis, and genes are ordered along high levels of expression typical of in vivo adipocytes. Further the y axis. The clusters are labeled A–AA, and boundaries between studies of the regulation of these and the other genes in this clusters are indicated by the alternating red and blue colorbar (far left). cluster should reveal whether a common regulatory mecha- Fold change relative to preconfluent 3T3-L1 cells is shown colorimetri- cally as indicated at the bottom left. Right, the normalized mean ex- nism underlies their high level of gene expression in vivo. pression level is shown for each cluster of genes in graphical form. 15 The expression analyses also revealed inconsistencies be- clusters of genes increased in abundance (left column), and 12 clusters tween adipogenesis in vitro and in vivo. aP2 has been consid- decreased in abundance during the course of differentiation (right ered to be a marker of the mature adipocyte in vitro because of column). Global Expression Profiles of Adipogenesis 34171 FIG.3. Transcription factors and sig- naling molecules changing in abun- dance during 3T3-L1 adipogenesis. A, the fold change relative to preconfluent ex- pression levels is shown for selected tran- scription factors changing in abundance during the course of adipogenesis and lipid deposition. These factors demonstrated sustained increases in expression through- out differentiation. B, other transcription factors were induced only transiently after confluence or the addition of growth fac- tors. A large group of transcription factors and signaling molecules decreased in abun- dance during differentiation (C and D)or decreased only transiently followed by a return to preconfluent or greater levels of expression (E). Only a few of these mole- cules had been identified previously as be- ing responsive to 3T3-L1 adipocyte differ- entiation. For reference, the cluster in which each factor was present Fig. 2 is indicated in superscript next to the gene name. DISCUSSION its dramatic induction during adipogenesis. The expression data revealed that aP2, although induced 2.7-fold in wild-type Oligonucleotide microarrays have been used to compare the mature adipocytes in vivo, was still expressed at high levels patterns of gene expression in preadipocytes and adipocytes in (80% of GAPDH expression) in preadipocytes in vivo. This vitro and in vivo. These analyses indicate that the gene expres- observation was verified independently by the Northern anal- sion changes associated with adipocyte development in vivo ysis of RNA from the adipocyte and stromal fractions of white and in vitro, although overlapping, are in many respects quite adipose tissue with a probe specific for the aP2 mRNA (data not different. Specifically, large groups of genes have been identi- shown). In contrast, PPAR2 and C/EBP, which are thought fied that are expressed at high levels in vivo and are not or to be responsible for the high level expression of aP2 in vitro, poorly expressed in 3T3-L1 cells. Additional noncell autono- are absent in preadipocytes in vivo and 40.9- and 31.3-fold mous factors may be necessary to achieve maximal levels of induced in vivo in adipocytes relative to background hybridiza- expression of these genes in vivo. Therefore, this study invokes tion intensities present in preadipocytes, respectively. This additional in vivo specific transcriptional programs as being apparent difference (i.e. why a target gene of PPAR2 and required for the development of the fully differentiated pheno- C/EBP is expressed in preadipocytes while the factors them- type of adipocytes in vivo. selves are absent in vivo) with the 3T3-L1 system remains to be The microarray data also indicated that the cellular pro- reconciled. grams associated with adipocyte differentiation are consider- 293 genes expressed in the stromal (preadipocyte) fraction ably more complex than previously appreciated and that a in vivo were not expressed or were expressed at very low number of previously uncharacterized gene regulatory events levels (Fig. 4C, Vivo2– 4) in both differentiated or undifferen- are likely to be activated during this process in vitro. Cluster tiated 3T3-L1 cells. Many molecules that have immune func- analysis of the 1249 genes that change in abundance during the tion were present in these clusters. Although some of these course of 3T3-L1 differentiation into adipocytes indicated that results could denote the presence of cell types other than the temporal pattern of gene expression can be described by at bona fide preadipocytes in this cell fraction, many of these least 27 distinct phases. These data emphasize the heretofore molecules including TNF, macrophage inflammatory pro- unappreciated complexity of the transcriptional programs ac- tein-2, IL-1b, IL-6, JE, KC, and C10-like chemokine were also tivated during adipogenesis and suggest the possibility that a expressed at 3–10-fold lower levels in adipocytes, suggest- larger number of genes than previously appreciated play a role ing that these immunologic cell markers were expressed in in this process. The complexity of these events was underesti- cells committed to the adipocyte lineage (Fig. 4C, Vivo2, and mated in a recent report in which the use of global expression Table I). These data are consistent with previous data from profiling identified genes differentially expressed between con- wild-type and ob/ob mice (20). fluent 3T3-L1 preadipocytes and day-6 differentiated 3T3-L1 Genes Enriched in Vitro—Finally, several groups of genes adipocytes (25). However, as indicated by the current analysis, that were expressed poorly in vivo but highly enriched in many clusters of genes that are transiently repressed or in- 3T3-L1 cells were evident (Fig. 4C, Vitro1–3, and Table I). duced during differentiation show equivalent expression levels These clusters of genes include PREF1, a marker of 3T3-L1 in confluent preadipocytes and day-7 adipocytes and would preadipocytes that is down-regulated during differentiation, but that has not been shown to be expressed in any cell type in have been missed by the previous report. The expression data generated from oligonucleotide microar- adipose tissue in vivo. These clusters of genes indicate addi- tional differences between 3T3-L1 cells and preadipocytes and rays verified the gene expression changes of many genes during adipocytes in vivo. adipocyte differentiation in 3T3-L1 cells including the tran- 34172 Global Expression Profiles of Adipogenesis phase that consists of 1–2 rounds of cell division prior to ter- minal differentiation. Prior to the addition of adipogenic fac- tors, cells are growth-arrested at confluence, indicated in this analysis by the potent repression of this large cluster of cell cycle genes (such as CDC25, centromere protein A, cyclin A, cyclin B, cyclin B1, cyclin B2, cytosolic thymidine kinase, to- poisomerase IIa, DNA ligase, DNA Pol catalytic subunit, CDC2, CDC20, CDK regulatory subunits 1 and 2, centromere protein A, inner centromere protein, mitotic centromere-asso- ciated kinesin, p34, CDC46, CDC47, ribonucleotide reductase M1, histone H2A.1, etc.) at the confluent and 6-h time points. However, after the addition of adipocyte-inducing factors that serves to induce cell division, this entire cluster of genes re- turns to preconfluent (dividing cell) expression levels at 12– 48 h. After this time, at which cells are known to have entered terminal differentiation, this entire cluster of genes is re- pressed permanently and dramatically. These expression pro- files provide new insight into the mRNA changes necessary for this phase of preadipocyte clonal expansion. Although the abil- ity of the adipogenic transcription factor PPAR to induce cell cycle arrest through the cyclin-dependent kinase inhibitors p18 and p21 has been demonstrated recently (28), the specific and additional events that lead to the regulation of this and other coordinated phases of gene repression remain to be explained. This unbiased approach toward expression characterization further implicated a number of previously unappreciated reg- ulatory molecules as playing a role in the development of the adipocyte phenotype in vitro. Regulation of RNA and protein levels of the known adipogenic transcription factors, e.g. C/EBP, C/EBP, C/EBP, PPAR, and SREBP-1, is not suffi- cient to generate the level of complexity seen during 3T3-L1 differentiation. For example, the kinetics of different known targets of these genes can in some cases be markedly different, e.g. PPAR targets aP2 and phosphoenolpyruvate carboxyki- nase. This observation suggests that many other genes either modulate the behavior of these known factors or act independ- ently to direct adipose gene expression. This study identifies a FIG.4. Comparison of preadipocyte and adipocyte gene ex- large number of such candidate regulatory molecules including pression levels in vivo and in vitro. 1435 genes that were enriched transcription factors, transcriptional coactivators, and signal- in preadipocytes or adipocytes were grouped by k-means clustering ing molecules. These data can now be analyzed further to test using a dot product metric according to their absolute level of expres- whether some of these factors can account for the regulation sion in 10 3T3-L1 time points and in isolated adipocytes and stromal cells from wild-type and ob/ob white adipose tissue. A, six clusters of evident in the clusters, the genes of which were expressed with genes labeled Adip1–Adip6 were enriched in adipocytes in vitro and in different kinetics from the clusters containing of PPAR, vivo and were expressed at quantitatively similar levels in those two C/EBP, SREBP-1, and their target genes. states. B, five clusters of genes labeled Preadip1–Preadip5 were more The relevance of these findings to adipogenesis in vivo was highly expressed in preadipocytes than cells that had accumulated cytoplasmic lipid both in vitro and in vivo. These clusters of genes evaluated further in a formal comparison of the expression provide novel markers for this unique population of preadipocyte cells. profile of in vitro preadipocytes and adipocytes to their in vivo C, four clusters of genes labeled Vivo1–Vivo4 were expressed at high counterparts. These data indicate that although adipocytes in levels in vivo and were undetectable or expressed at much lower levels vitro express many of the same genes as well as morphologic in vitro. One of these clusters, Vivo1, is enriched specifically in adipo- cytes in vivo and is lowly or not expressed in vitro. In cluster Vivo2, and metabolic characteristics of in vivo adipocytes, the expres- many genes were expressed in cells of the preadipocyte/adipocyte line- sion profile of preadipocytes and adipocytes in vitro are differ- age that were not expressed in vitro. In the clusters Vivo3 and Vivo4, ent from those of the stromal and adipocyte fractions of white genes were uniquely expressed in the stromal fraction isolated from adipose tissue in vivo. The key lipogenic enzymes ATP-citrate wild-type and ob/ob adipose tissue. These two clusters characterize the population of in vivo cells that includes preadipocytes and possibly lyase and phosphoenolpyruvate carboxykinase are expressed other cell types responsible for supporting the fully differentiated adi- at 20 –200-fold lower levels (at day 7) or 2–10-fold lower levels pocyte phenotype. Three clusters of genes labeled Vitro1–Vitro3 were (at day 28) in vitro relative to in vivo adipocytes, suggesting at more highly expressed in vitro and expressed at low or undetectable least one possible explanation for the lower total accumulation levels in vivo. of triglyceride in cultured adipocytes relative to in vivo adipo- scription factors C/EBP, PPAR2, SREBP-1, C/EBP, cytes. These results suggest that other heretofore unknown factors are necessary for the development of the fully differen- C/EBP, CHOP-10, AEBP1, COUP-TF (4, 26, 27), and others (see arrays.rockefeller.edu/obesity/adipocyte for the complete tiated adipocyte in vivo. The data also suggest that preadipo- cytes in vivo exhibit a novel phenotype that is not entirely list of differentially expressed molecules). A large group of genes was repressed during in vitro adipo- mimicked by preadipocytes in vitro. These cells express a num- genesis. One particular phase of interest is a large group of cell ber of genes with immune functions, suggesting an even cycle-related genes (Fig. 2R). 3T3-L1 cells, after the addition of broader array of roles for the in vivo preadipocyte than previ- growth factors at confluence, go through a clonal expansion ously appreciated. Global Expression Profiles of Adipogenesis 34173 TABLE I Genes responsive to adipocyte differentiation in vivo and in vitro Genes from adipocyte-specific (common to in vivo and in vitro), preadipocyte-specific (common to in vitro and in vivo), in vivo specific, and in vitro specific clusters are shown along with the cluster they were present in from Fig. 4. Transcription factors are shown in bold type. Complete membership of these clusters is available online at arrays.rockefeller.edu/obesity/adipocyte. In vivo and in vitro adipocyte In vivo and in vitro preadipocyte In vivo enriched genes In vitro enriched genes enriched genes enriched genes Gene Cluster Gene Cluster Gene Cluster Gene Cluster ACRP30/AdipoQ/Adiponectin Adip1 HMGl-Y Preadip1 ATP-Citrate Lyase Vivo1 PREF1 Vitro1 Adipsin Adip1 IFN Preadip1 Caveolin-1 Vivo1 Osteoblast Spec. 1 Vitro1 Aldehyde DH Adip1 Nuclear LIM Preadip1 PEPCK Vivo1 Mdm2 Vitro1 interactor Angiotensinogen Adip1 Prx2 Homeobox Preadip1 Acetyl-CoA Synthetase Vivo1 mrp/plf3 proliferin Vitro1 aP2 Adip1 TSC-36 Preadip1 Frizzled4 Vivo1 Annexin VIII Vitro1 Fat Specific Protein 27 Adip1 -Amylase Preadip2 Skeletal LIM FHL 1 Vivo1 Id HLH Factor Vitro1 Glycerophosphate DH Adip1 a-B2 Crystallin Preadip2 17-hydroxysteroid DH Vivo1 Apoptosis signal-reg Vitro2 kinase1 Haptoglobin Adip1 AEBP1 Preadip2 High MW GH Receptor Vivo1 Sox-4 Vitro2 Hormone Sensitive Lipase Adip1 Id related Preadip2 TSH Receptor Vivo1 AP-2 Vitro2 LAF1 Transketolase Adip1 junB Preadip2 obese mRNA Vivo1 FK506BP-13 Vitro2 Long Chain Fatty Acyl-CoA Adip1 Kruppel-like factor Preadip2 TNF Vivo2 Galactokinase Vitro2 Synthetase Transferrin Adip1 MCSF Preadip2 Zn Finger A20 Vivo2 p160 myb BP Vitro2 ACTH Receptor Adip2 PAI-1 Preadip2 SOCS-3 Vivo2 Proliferin Vitro2 Acyl-CoA BP Adip2 Spi2 Preadip2 C1q a-chain Vivo2 FK506BP-51 Vitro2 Angiotensinogen Adip2 FGF Receptor Preadip3 C1q b-chain Vivo2 HMGI-C Vitro2 ApoC1 Adip2 Forkhead Box F2 Preadip3 C1q c-chain Vivo2 cyclin G Vitro3 3 Adrenoceptor Adip2/3 Histone Deacetylase Preadip3 PDGF-inducible JE Vivo2 FK506BP-65 Vitro3 HD1 C/EBP Adip2 LCAT Preadip3 PDGF-inducible KC Vivo2 Zn Finger HRX/ Vitro3 ALL-1 CPT II Adip2 Transcription Preadip3 MIP-2 Vivo2 Keratinocyte Growth Vitro3 Factor E2a Factor Citrate Transporter Adip2/3 Zn Finger Zic3 Preadip3 IL-1b Vivo2 Frizzled Vitro3 GLUT-4 Adip2 c-myc Preadip4 IL-6 Vivo2 SOCS-2 Vitro3 Insulin Activated AA Adip2 C/EBP Preadip4 c-fos Vivo2 Pbx3b Vitro3 Transporter Malate DH Adip2 Endothelial MAP1 Preadip4 fosE Vivo2 C/EBP Vitro3 Malic Enzyme Adip2 TNF-Receptor-1 Preadip4 IP-10 Cytokine Vivo2 MEST Adip2 Erythroid Kruppel- Preadip4 Zn Finger p36 Vivo2 like Monoglyceride Lipase Adip2 204 interferon Preadip5 zif/268/Krox24 Vivo2 activatable p18 Adip2 ApoD Preadip5 LRG-21 Vivo2 p19 Adip2 fos-related antigen Preadip5 C10-like Chemokine Vivo2 PPAR2 Adip2 HES-1 HLH Preadip5 Gut Enriched Kruppel- Vivo2 like Spot14 Adip2 Mbh-1 Preadip5 Krox-20 Vivo2 SREBP-1 Adip2 Mkr3 Zn Finger Preadip5 ICAM-1 Vivo2 Glucose-6-P DH Adip3 OSF-2 Preadip5 RDC1 Chemokine Receptor Vivo3/4 Histone H1 Adip3 p85 Secreted Preadip5 junB Vivo3 TOR/ROR Adip3 PDGF Receptor Preadip5 Enkephalin Vivo3 VLDL Receptor Adip3 Prostacyclin Preadip5 Cathepsin S Vivo3 Synthase FPP Synthetase Adip3 EF1 Preadip5 Cathepsin H Vivo3 3-Phosphogylcerate DH Adip4 UCP-2 Preadip5 CXCR-4 Vivo3 Fatty Acid Synthase Adip4 VCAM-1 Preadip5 IL-1 Vivo3 Low MW GH Receptor Adip4 PAI-2 Vivo3 PFK-1 Adip4 PU.1 Vivo3 Mal1 Lipid Binding Adip4 C10 Vivo3 Pyruvate Carboxylase Adip4 Cor1 Basic HLH Factor Vivo4 RXRa Adip4 Cytokine Receptor-like EBI3 Vivo4 SCD1 Adip4 Prostaglandin E Receptor Vivo4 Transaldolase Adip4 PECAM-1 Vivo4 Triosephosphate Isomerase Adip4 EBI-1 G-protein Receptor Vivo4 VEGF Adip4 TRAF-1 Vivo4 VEGF-b Adip4 IL-3 Receptor Vivo4 SCD2 Adip5 Angiogenin Vivo4 Lactate DH Adip5 Endothelin B Receptor Vivo4 Enolase Adip5/6 Macrophage Specific NRAMP Vivo4 PHAS II Adip5 Macrophage Metalloelastase Vivo4 Cyclin D Adip5 MIP-1 Vivo4 Cyclin E Adip5 Neutrophil Cytosol Factor2 Vivo4 3-hydroxyacyl-CoA DH Adip6 IL-4 Receptor Vivo4 Adipocyte p27 Adip6 IL-10 Vivo4 CHOP-10 Adip6 RANTES Vivo4 Cytosolic Malate DH Adip6 Lymphoid Spec. IFN Reg. Vivo4 Fact Dihydrolipoamide DH Adip6 Nurr1 Nuclear Orphan Vivo4 Receptor p21/Waf1 Adip6 Prostaglandin Synthase Vivo4 Aldolase A Adip6 34174 Global Expression Profiles of Adipogenesis 1108 –1112 These in vivo expression data provide a new framework in 12. Tanaka, T., Yoshida, N., Kishimoto, T., and Akira, S. (1997) EMBO J. 16, which the functional significance of observed changes in vari- 7432–7443 13. Rosen, E. D., Sarraf, P., Troy, A. E., Bradwin, G., Moore, K., Milstone, D. S., ous in vitro models of adipogenesis can be evaluated. Finally, Spiegelman, B. M., and Mortensen, R. M. (1999) Mol. Cell 4, 611– 617 these data suggest the importance of evaluating cell culture 14. Barak, Y., Nelson, M. C., Ong, E. S., Jones, Y. Z., Ruiz-Lozano, P., Chien, K. R., models for studying in vivo phenomena using microarrays. Koder, A., and Evans, R. M. (1999) Mol. Cell 4, 585–595 15. 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Distinct Transcriptional Profiles of Adipogenesisin Vivo and in Vitro

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 36, Issue of September 7, pp. 34167–34174, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Distinct Transcriptional Profiles of Adipogenesis in Vivo and in Vitro* Received for publication, May 15, 2001 Published, JBC Papers in Press, July 9, 2001, DOI 10.1074/jbc.M104421200 Alexander Soukas‡, Nicholas D. Socci§¶, Barbara D. Saatkamp‡, Silvia Novelli‡, and Jeffrey M. Friedman‡** From the ‡Laboratory of Molecular Genetics, the §Center for Studies in Physics and Biology, and the Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, and the ¶Institute of Theoretical Physics, University of California, Santa Barbara, California 93106 Obesity, defined as an increase in adipose tissue mass, esis is the result of a temporally ordered pattern of 3–5 distinct is the most prevalent nutritional disorder in industrial- phases of gene expression (summarized in the above reviews). ized countries and is a growing problem in developing Indeed several of the more than 100 molecules that have been countries. An increase in adipose tissue mass can be the identified as differentially expressed during the transition of result of the production of new fat cells through the preadipocytes to adipocytes in culture have been shown to have process of adipogenesis and/or the deposition of in- binding sites for the transcription factors PPAR and C/EBP creased amounts of cytoplasmic triglyceride per cell. in their promoters (9, 10). Although much has been learned about the differentia- Although cell culture models of adipocytes faithfully express tion of adipocytes in vitro, less is known about the mo- many genes that are markers of adipocytes in vivo, the events lecular basis for the mechanisms regulating adipogene- that trigger this transformation in vivo are not as well under- sis in vivo. Here oligonucleotide microarrays have been stood. Knockout studies of C/EBP, C/EBP, C/EBP, and used to compare the patterns of gene expression in prea- PPAR have confirmed that these molecules are necessary in dipocytes and adipocytes in vitro and in vivo. These data vivo for adipogenesis, but it is not known with certainty indicate that the cellular programs associated with adi- whether these factors are sufficient (11–15). Thus it is unclear pocyte differentiation are considerably more complex whether the high level of expression of these factors evident in than previously appreciated and that a greater number in vitro adipocytes can recapitulate the gene expression profile of heretofore uncharacterized gene regulatory events of adipocytes in vivo. It has already been shown that some are activated during this process in vitro. In addition, adipocyte-derived molecules such as leptin are expressed at the gene expression changes associated with adipocyte lower levels in cultured adipocytes (16) and that in vivo levels development in vivo and in vitro, while overlapping, are in some respects quite different. These data further sug- of ob mRNA are restored in fat pads derived from subcutane- gest that one or more transcriptional programs are ac- ously implanted 3T3-F442A preadipocytes (17). tivated exclusively in vivo to generate the full adipocyte To characterize the regulation of gene expression during phenotype. This gene expression survey now sets the adipogenesis in vivo and in vitro further, the abundance of stage for further studies to dissect the molecular differ- 11,000 genes and expressed sequence tags was measured at 10 ences between in vivo and in vitro adipocytes. different time points during in vitro 3T3-L1 adipocyte differen- tiation using oligonucleotide microarrays (Affymetrix, Santa Clara, CA). The abundance of the same 11,000 genes was also Adipogenesis has been studied extensively in vitro using a measured in adipocytes and stromal cells (including preadipo- number of preadipocyte cell lines including 3T3-L1 cells (1). cytes) isolated from wild-type and ob/ob white adipose tissue. When cultured in defined media, 3T3-L1 cells deposit triglyc- Independent analyses of these data indicated that a more com- eride in cytoplasmic lipid droplets and express genes that are plex program of gene expression than was known previously is also expressed in adipocytes in vivo (2– 8). Elegant studies of activated during adipocyte differentiation in vitro and in vivo. this process have led to the identification of several key regu- Comparative analysis of the in vitro and in vivo expression latory genes that are necessary and/or sufficient for the tran- data revealed that although some genes are expressed at in sition of preadipocytes into adipocytes in vitro including vivo levels in fully differentiated 3T3-L1 cells, most notably CCAAT/enhancer binding proteins (C/EBPs) , , and  and target genes for C/EBP and PPAR , large clusters of genes peroxisome proliferator-activated receptor (PPAR)  (9). Stud- are expressed at much higher levels or even exclusively in vivo. ies of these transcription factors have suggested that adipogen- Conversely, a large group of genes is expressed in vitro that is not or is poorly expressed in vivo in adipocytes or preadipo- cytes. Although these data represent a descriptive survey of * This work was supported by NINDS National Institutes of Health Grant NS39662 (to J. M. F and N. D. S), MSTP National Institutes of gene expression profiles of adipogenesis in vivo and in vitro, Health Grant GM07739 (to A. S.) and by NSF National Institutes of together they indicate that adipogenesis is likely to be more Health Grant PHY99-07949 (to N. D. S). The costs of publication of this complex than previously appreciated and that specific tran- article were defrayed in part by the payment of page charges. This scriptional programs that generate the full adipocyte pheno- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. type are not activated in cultured adipocytes but are activated ** To whom correspondence should be addressed: The Rockefeller by signals present exclusively in vivo. University, 1230 York Ave., Box 305, New York, NY 10021. Tel.: 212- 327-8800; Fax: 212-327-7420; E-mail: [email protected]. EXPERIMENTAL PROCEDURES The abbreviations used are: C/EBP, CCAAT/enhancer binding pro- 3T3-L1 Cell Culture—3T3-L1 cells (ATCC, Manassas, VA) were tein; PPAR, peroxisome proliferator-activated receptor; SREBP, sterol regulatory element-binding protein. maintained in subconfluent cultures in Dulbecco’s modified Eagle’s This paper is available on line at http://www.jbc.org 34167 This is an Open Access article under the CC BY license. 34168 Global Expression Profiles of Adipogenesis medium supplemented with 33 M biotin, 17 M calcium pantothenate, The initial analysis of the expression data from the oligonu- 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomy- cleotide arrays indicated that the abundance of 1259 genes cin, and 0.25 g/ml amphotericin B (Life Technologies, Inc.). For differ- changed 3-fold or more during the course of the differentiation entiation, 3  10 cells were seeded per 100-mm plate, allowed to reach response. These data corroborated many previously reported 100% confluence, and induced 1 day post confluence with the above patterns of gene expression including changes of PREF1, M insulin, 2 nM triiodothyronine, 250 medium supplemented with 170 n nM dexamethasone, and 500 M isobutylmethylxanthine for 2 days (Life AEBP1, C/EBP, C/EBP, C/EBP, aP2, adipsin, Acrp30/Adi- Technologies, Inc.). After induction, the cells were fed every 2 days with poQ/adiponectin, lipoprotein lipase, hormone-sensitive lipase, maintenance medium supplemented with 170 nM insulin and 2 nM T3. stearoyl-CoA desaturase 1, 2 type VI collagen RNAs, and At indicated time points (Fig. 1), the medium was drained, the cells others (see below). were lysed by the addition of Trizol reagent (Life Technologies, Inc.), These data were analyzed using a modified k-means cluster- and total RNA was isolated as per protocol. Northern blotting was ing algorithm with a dot product metric, which groups genes conducted as described previously (18). For histology, cells were grown and differentiated as described above on lab-tek slides (Nunc, Naper- based on the similarity of their patterns of gene expression ville, IL), fixed (2% formaldehyde/0.2% glutaraldehyde in PBS for 15 (20). Cluster analysis indicated that the 1259 differentially min.), rinsed in PBS, stained with oil-red O (0.7% in 60% isopropanol for expressed genes can be grouped most parsimoniously into 27 10 min), and counter-stained with hematoxylin (40% in water for 5 temporally distinct patterns, each containing between 16 and min). 118 genes (Fig. 2), suggesting that the regulation of adipogen- Adipose Tissue Fractionation—Adipose tissue was divided into stro- esis may be considerably more complex than previously appre- mal and adipocyte fractions as described previously (19). Briefly, freshly excised peri-uterine fat pads from 8-week-old female C57Bl/6J, ciated. These clusters include 1000 genes not identified pre- wild-type, or ob/ob mice were rinsed in PBS, minced, and digested for 45 viously as being differentially regulated during adipocyte min–1hat37 °C in Krebs-Ringer bicarbonate (pH 7.4) with 4% bovine differentiation and more than 100 known transcription factors serum albumin and 1.5 mg/ml type I collagenase (Worthington, Free- and signaling molecules. hold, NJ). The digested tissue was filtered through a 250-m nylon 15 distinct clusters of RNAs increased in abundance during mesh to remove undigested tissue and centrifuged at 500  g for 5 min. adipogenesis in vitro (Fig. 2, left). 12 clusters of genes de- The floating adipocyte fraction was removed, washed in buffer, and recentrifuged to isolate free adipocytes. The stromal-vascular pellet was creased during adipogenesis (Fig. 2, right). Because of space resuspended in erythrocyte lysis buffer (154 mM NH4Cl, 10 mM restrictions, only a subset of the genes identified by this anal- KHCO3, and 1 mM EDTA), filtered through a 28-m nylon mesh to ysis are shown. All of the data are available online (arrays. remove endothelial cells, and pelleted at 500  g for 5 min. Total RNA rockefeller.edu/obesity/adipocyte). from the adipocyte and stromal fractions was isolated with Trizol rea- gent from five different ob/ob and wild-type preparations from which equal amounts were pooled for microarray analysis. Adequate separa- Patterns of Gene Expression in Adipocyte Differentiation tion of adipocyte and stromal fractions was confirmed by Northern Invoke Multiple Additional Transcriptional Mechanisms blotting for the adipocyte markers aP2 and PPAR (data not shown). Affymetrix Oligonucleotide Microarray Analysis and k-means Clus- Induced Genes—Microarray analysis indicated that the tran- tering—Samples were prepared for murine 11k microarrays from 10 g scription factors SREBP-1 (Fig. 2, A and C), C/EBP (Fig. 2C), of total RNA as outlined in the Affymetrix technical bulletin and as and PPAR2 (Fig. 2E) were up-regulated dramatically, each described previously (20). Hybridization and analysis were carried out with different kinetics, over the course of adipocyte differenti- using Affymetrix hybridization, washing, scanning, and Genechip 3.3 ation. Many genes that are markers of the differentiated adi- analysis stations as described in the Affymetrix technical manual. For 3T3-L1 analysis clustering, genes were included if greater than 3-fold pocyte increased in parallel with these factors. These included and more than 500 average difference change units (abundance meas- many known gene targets of these factors including the urement) increased or decreased in one time point relative to precon- SREBP-1 and C/EBP target genes fatty acid synthase (Fig. fluent expression levels. These genes were clustered according to fold 2C), stearoyl-CoA desaturase-1 (Fig. 2C), stearoyl-CoA desatu- change value using a modified k-means clustering algorithm with a dot rase-2 (Fig. 2D), the PPAR and C/EBP target gene aP2 (Fig. product metric described previously (20). For comparative in vivo and in 2E), and the highly adipocyte-enriched genes glycerophosphate vitro adipocyte and preadipocyte cluster analysis, genes were filtered to include only those genes that were either 5-fold enriched in in vivo dehydrogenase (Fig. 2E), adipsin (Fig. 2D), and Acrp30/Adi- adipocytes or preadipocytes (wild-type or ob/ob)or in vitro 3T3-L1 cells poQ/adiponectin (21) (Fig. 2D). at any time point relative to liver, hypothalamus, skeletal muscle, or However, many genes that mark the differentiated adipocyte pancreas. Genes were also included that were more than 5-fold different were expressed with different kinetics than SREBP-1, C/EBP, between in vivo preadipocytes and adipocytes (wild-type and ob/ob). and PPAR. For example, phosphoenolpyruvate carboxyki- Raw expression levels (average difference) in in vivo and in vitro sam- nase, a glycerogenic enzyme that has been shown to have a ples were set to 100 if they were below this value (to eliminate con- founding effects of background variations), log-transformed, baseline PPAR binding site in its promoter (22), is first expressed in normalized, and clustered (according to absolute abundance/hybridiza- adipocytes at 7 days and continues to increase in abundance tion intensity) with the above modified k-means clustering algorithm to until 28 days (Fig. 2G). The 3-adrenergic receptor, cytochrome group genes with similar patterns of expression. c oxidase VIIIH, glucose-6-phosphate isomerase, phosphofruc- tokinase I, and insulin-like growth factor II demonstrate a RESULTS similar pattern of expression and are also in this cluster. These 3T3-L1 Adipocyte Differentiation Occurs Through Multiple, patterns differed from the expression profile of the aforemen- Overlapping, Coordinated Phases of Gene Expression tioned transcription factors. These observations suggest that RNA was prepared from preconfluent and confluent preadi- other regulatory factors also play a role in adipogenesis. pocytes and from cells 6, 12, 24, and 48 h and 3, 4, 7, and 28 Repressed Genes—12 clusters of genes including cell cycle days after the cells were induced to the differentiate (Fig. 1A). genes (Fig. 2, Q and R), cytoskeletal genes (Fig. 2, Q and T), A standard differentiation protocol was optimized to maximize splicing factors and protein turnover genes (Fig. 2U), and the percentage of 3T3-L1 cells accumulating cytoplasmic lipid markers of other cell types including myelocytes and lympho- (greater than 90% at 7–28 days). Histologic staining for cyto- cytes (Fig. 2S) were down-regulated during differentiation. plasmic lipid confirmed that the cells had fully differentiated These clusters decrease with varying kinetics beginning as (Fig. 1A). Northern blotting with probes specific for aP2 and early as confluence (Fig. 2, P, R, W, X, and AA), after the PPAR, markers of mature adipocytes, confirmed that these addition of growth factors (Fig. 2Q), or later in the process of genes were expressed at in vivo levels in the differentiated cells adipocyte maturation (Fig. 2, S, T, U, V, Y, and Z). Although (Fig. 1B). several clusters contained transcription factors known to be Global Expression Profiles of Adipogenesis 34169 FIG.1. Time-course, histologic, and Northern analysis of 3T3-L1 differen- tiation. A, 3T3-L1 cells were seeded into 100-mm dishes at 3  10 cells/plate, and RNA was harvested at 10 points (indicat- ed by arrows) before or after initiation of differentiation by the addition of growth factors. Each of these samples was used for oligonucleotide microarray analysis. Histologic staining for lipid using oil-red O indicated that cells had begun to accu- mulate lipid by 3 days and had large cen- tral lipid droplets by 7–14 days. B, North- ern blotting for aP2 and PPAR of 10 g of total RNA indicated that the 3T3-L1 cells differentiated as described previ- ously (left panels) and expressed adipo- cyte genes at quantitatively similar levels relative to in vivo adipocytes (right pan- els). WT, wild type. repressed during adipogenesis including COUP-TF1 (Fig. 2Q) even C/EBP (up 3.6-fold) and C/EBP (up 3.9-fold) (Fig. 3B), and AEBP1 (Fig. 2Y), little is known about the transcriptional which have been invoked previously as responsible for mediat- mechanisms responsible for regulating these clusters of genes. ing the early phases of adipogenesis (23). All of these factors Transcription Factors and Signaling Molecules Regulated are transiently up-regulated after the addition of growth fac- During Adipogenesis—More than 100 known additional tran- tors to stimulate adipose conversion of confluent adipocytes. scription factors, transcriptional coactivators, and signaling Thyroid hormone receptor c-erbA-2 increased 13.8-fold prior molecules were regulated during adipogenesis. These factors to the addition of growth factors and remained elevated were either stably induced or repressed during differentiation throughout differentiation. Together, these results suggest the (Fig. 3, A, C, and D) or transiently regulated (Fig. 3, B and E). possible involvement of a large cluster of regulatory molecules In addition, in all clusters where a known adipogenic transcrip- and signaling pathways during differentiation. tion factor was present, additional transcription factors were Gene expression analyses indicated that a large group of coexpressed. Coordinate with a 71-fold increase in C/EBP and DNA binding inhibitors (Id genes) and high mobility group a 20.6-fold increase in SREBP-1, the transcription factors X- proteins (HMG genes) are down-regulated with distinct kinetic box-binding protein (up 6.4-fold), estrogen receptor-related  profiles during differentiation (Fig. 3C) These gene regulatory (up 4.2-fold), and Ig/enhancer binding protein (up 5.2-fold) were changes are accompanied by the transient or sustained down- up-regulated (Figs. 3A and 2C). The 18.1-fold induction of regulation of fos/jun family members, homeobox, forkhead, and PPAR was associated with a 15.3-fold up-regulation of the other transcription factors (Fig. 3, D and E). The role these transcriptional corepressor RIP140 and a 14.6- and 3.3-fold complex patterns play in generation of the differentiated adi- up-regulation of the transcription factors STAT-1 and iron pocyte remains to be determined. response element binding protein, respectively (Figs. 3A and Gene Expression in Adipocytes and Preadipocytes in Vivo 2E). In addition to these factors, which show similar profiles to C/EBP, SREBP-1, and PPAR, other transcription factors The phenotype of preadipocytes and adipocytes in vitro and were identified, which show different transcriptional profiles in vivo was compared by scoring the abundance of the same including Mxi-1, Zic3, and the albumin D-box-binding protein. 11,000 genes in RNA from the adipocyte and stromal (preadi- The glucocorticoid-induced leucine zipper (up 42.7-fold), N10 pocyte) fractions of C57Bl/6 wild-type and ob/ob mice. A total of nuclear hormone receptor (up 11.8-fold), and Wnt-4 signaling 1435 genes represented on the array were at least 5-fold en- molecule (up 34.8-fold) were induced to a greater extent than riched in adipocytes or preadipocytes as compared with other 34170 Global Expression Profiles of Adipogenesis tissues including liver, brain, skeletal muscle, and exocrine pancreas (see “Experimental Procedures”). k-means clustering of the absolute expression levels of these genes in adipocytes and preadipocytes in vivo and in vitro identified 18 distinct groupings of genes with significant expression levels. These included six clusters of genes that were enriched in adipocytes both in vivo and in vitro, five clusters of genes that were enriched in preadipocytes in vivo and in vitro, four clusters of genes that were specifically expressed in vivo, and three clus- ters that were expressed specifically in 3T3-L1 cells (Fig. 4). Genes Expressed in Vivo and in Vitro—This analysis con- firmed that for many genes, differentiated 3T3-L1 cells accu- rately recapitulate the in vivo patterns of gene expression observed in the transition of preadipocytes to adipocytes (Fig. 4A). Thus, many genes that are not expressed (or expressed at a low level) in preadipocytes were highly expressed in mature 3T3-L1 adipocytes, wild-type adipocytes in vivo, and ob/ob adi- pocytes in vivo (Table I). These six clusters of adipocyte-en- riched genes varied principally in their absolute level of expres- sion and were further divided into distinct subgroups by the k-means algorithm. The most notable members of this group include C/EBP, PPAR2, SREBP-1 (nonspecific SREBP-1a/1c probe set), and RXR. Many other adipocyte-specific and adi- pocyte-enriched genes are present in these groups including genes necessary to synthesize fatty acids from acetyl-CoA, the GLUT-4 glucose transporter, aP2, ACRP30/AdipoQ/adiponec- tin, and the 3-adrenergic receptor (Table I). A second group of five clusters were highly expressed in preadipocytes in vitro and in vivo and decreased in abundance during adipocyte conversion (Fig. 4B). These clusters include the transcriptional repressor AEBP1, which is negatively reg- ulated during adipogenesis and in preadipocytes serves to neg- atively regulate the aP2 AE-1 enhancer (24). The transcription factors Prx2 homeobox, junB, nuclear LIM interactor, Kruppel- like factor, forkhead box F2, E2a, and C/EBP are among others highly expressed in preadipocytes (prior to the accumu- lation of cytoplasmic lipid) and down-regulated during differ- entiation (Table I). Genes Expressed at High Levels in Vivo and Absent or Lower Levels in Vitro—68 genes that were highly expressed in adipo- cytes in vivo were expressed at an average of 20-fold lower levels in differentiated 3T3-L1 adipocytes (Fig. 4C, Vivo1). This cluster of genes indicates that adipocytes in vitro do not express the fully differentiated in vivo phenotype. This group of in vivo enriched genes included some genes encoding metabolic en- zymes such as ATP-citrate lyase (10.4-fold lower in 3T3-L1 day-28 adipocytes than wild-type adipocytes), phosphoenol- pyruvate carboxykinase (200-fold lower on day 7 and 1.9-fold lower on day 28), acetyl-CoA synthetase (3.1-fold lower on day 28), and leptin mRNA (63.4-fold lower on day 28). The high molecular weight growth hormone receptor and the thyrotropin receptor are 5.5- and 6.1-fold more highly expressed in vivo. Finally, the transcription factor skeletal muscle LIM protein FHL1 is 18.4-fold more highly expressed in wild-type adipo- cytes relative to 3T3-L1 day-28 adipocytes, which show only background hybridization levels. These results indicate that FIG.2. k-means cluster analysis of genes changing in abun- the absolute levels of expression of these genes are lower in dance during 3T3-L1 differentiation into adipocytes. Left, 1259 adipocytes in vitro, and although some are induced during genes were grouped into 27 clusters according to their profile of expres- sion across 10 time points by a modified k-means cluster algorithm. differentiation, additional signals seem necessary to direct Experiments are ordered along the x axis, and genes are ordered along high levels of expression typical of in vivo adipocytes. Further the y axis. The clusters are labeled A–AA, and boundaries between studies of the regulation of these and the other genes in this clusters are indicated by the alternating red and blue colorbar (far left). cluster should reveal whether a common regulatory mecha- Fold change relative to preconfluent 3T3-L1 cells is shown colorimetri- cally as indicated at the bottom left. Right, the normalized mean ex- nism underlies their high level of gene expression in vivo. pression level is shown for each cluster of genes in graphical form. 15 The expression analyses also revealed inconsistencies be- clusters of genes increased in abundance (left column), and 12 clusters tween adipogenesis in vitro and in vivo. aP2 has been consid- decreased in abundance during the course of differentiation (right ered to be a marker of the mature adipocyte in vitro because of column). Global Expression Profiles of Adipogenesis 34171 FIG.3. Transcription factors and sig- naling molecules changing in abun- dance during 3T3-L1 adipogenesis. A, the fold change relative to preconfluent ex- pression levels is shown for selected tran- scription factors changing in abundance during the course of adipogenesis and lipid deposition. These factors demonstrated sustained increases in expression through- out differentiation. B, other transcription factors were induced only transiently after confluence or the addition of growth fac- tors. A large group of transcription factors and signaling molecules decreased in abun- dance during differentiation (C and D)or decreased only transiently followed by a return to preconfluent or greater levels of expression (E). Only a few of these mole- cules had been identified previously as be- ing responsive to 3T3-L1 adipocyte differ- entiation. For reference, the cluster in which each factor was present Fig. 2 is indicated in superscript next to the gene name. DISCUSSION its dramatic induction during adipogenesis. The expression data revealed that aP2, although induced 2.7-fold in wild-type Oligonucleotide microarrays have been used to compare the mature adipocytes in vivo, was still expressed at high levels patterns of gene expression in preadipocytes and adipocytes in (80% of GAPDH expression) in preadipocytes in vivo. This vitro and in vivo. These analyses indicate that the gene expres- observation was verified independently by the Northern anal- sion changes associated with adipocyte development in vivo ysis of RNA from the adipocyte and stromal fractions of white and in vitro, although overlapping, are in many respects quite adipose tissue with a probe specific for the aP2 mRNA (data not different. Specifically, large groups of genes have been identi- shown). In contrast, PPAR2 and C/EBP, which are thought fied that are expressed at high levels in vivo and are not or to be responsible for the high level expression of aP2 in vitro, poorly expressed in 3T3-L1 cells. Additional noncell autono- are absent in preadipocytes in vivo and 40.9- and 31.3-fold mous factors may be necessary to achieve maximal levels of induced in vivo in adipocytes relative to background hybridiza- expression of these genes in vivo. Therefore, this study invokes tion intensities present in preadipocytes, respectively. This additional in vivo specific transcriptional programs as being apparent difference (i.e. why a target gene of PPAR2 and required for the development of the fully differentiated pheno- C/EBP is expressed in preadipocytes while the factors them- type of adipocytes in vivo. selves are absent in vivo) with the 3T3-L1 system remains to be The microarray data also indicated that the cellular pro- reconciled. grams associated with adipocyte differentiation are consider- 293 genes expressed in the stromal (preadipocyte) fraction ably more complex than previously appreciated and that a in vivo were not expressed or were expressed at very low number of previously uncharacterized gene regulatory events levels (Fig. 4C, Vivo2– 4) in both differentiated or undifferen- are likely to be activated during this process in vitro. Cluster tiated 3T3-L1 cells. Many molecules that have immune func- analysis of the 1249 genes that change in abundance during the tion were present in these clusters. Although some of these course of 3T3-L1 differentiation into adipocytes indicated that results could denote the presence of cell types other than the temporal pattern of gene expression can be described by at bona fide preadipocytes in this cell fraction, many of these least 27 distinct phases. These data emphasize the heretofore molecules including TNF, macrophage inflammatory pro- unappreciated complexity of the transcriptional programs ac- tein-2, IL-1b, IL-6, JE, KC, and C10-like chemokine were also tivated during adipogenesis and suggest the possibility that a expressed at 3–10-fold lower levels in adipocytes, suggest- larger number of genes than previously appreciated play a role ing that these immunologic cell markers were expressed in in this process. The complexity of these events was underesti- cells committed to the adipocyte lineage (Fig. 4C, Vivo2, and mated in a recent report in which the use of global expression Table I). These data are consistent with previous data from profiling identified genes differentially expressed between con- wild-type and ob/ob mice (20). fluent 3T3-L1 preadipocytes and day-6 differentiated 3T3-L1 Genes Enriched in Vitro—Finally, several groups of genes adipocytes (25). However, as indicated by the current analysis, that were expressed poorly in vivo but highly enriched in many clusters of genes that are transiently repressed or in- 3T3-L1 cells were evident (Fig. 4C, Vitro1–3, and Table I). duced during differentiation show equivalent expression levels These clusters of genes include PREF1, a marker of 3T3-L1 in confluent preadipocytes and day-7 adipocytes and would preadipocytes that is down-regulated during differentiation, but that has not been shown to be expressed in any cell type in have been missed by the previous report. The expression data generated from oligonucleotide microar- adipose tissue in vivo. These clusters of genes indicate addi- tional differences between 3T3-L1 cells and preadipocytes and rays verified the gene expression changes of many genes during adipocytes in vivo. adipocyte differentiation in 3T3-L1 cells including the tran- 34172 Global Expression Profiles of Adipogenesis phase that consists of 1–2 rounds of cell division prior to ter- minal differentiation. Prior to the addition of adipogenic fac- tors, cells are growth-arrested at confluence, indicated in this analysis by the potent repression of this large cluster of cell cycle genes (such as CDC25, centromere protein A, cyclin A, cyclin B, cyclin B1, cyclin B2, cytosolic thymidine kinase, to- poisomerase IIa, DNA ligase, DNA Pol catalytic subunit, CDC2, CDC20, CDK regulatory subunits 1 and 2, centromere protein A, inner centromere protein, mitotic centromere-asso- ciated kinesin, p34, CDC46, CDC47, ribonucleotide reductase M1, histone H2A.1, etc.) at the confluent and 6-h time points. However, after the addition of adipocyte-inducing factors that serves to induce cell division, this entire cluster of genes re- turns to preconfluent (dividing cell) expression levels at 12– 48 h. After this time, at which cells are known to have entered terminal differentiation, this entire cluster of genes is re- pressed permanently and dramatically. These expression pro- files provide new insight into the mRNA changes necessary for this phase of preadipocyte clonal expansion. Although the abil- ity of the adipogenic transcription factor PPAR to induce cell cycle arrest through the cyclin-dependent kinase inhibitors p18 and p21 has been demonstrated recently (28), the specific and additional events that lead to the regulation of this and other coordinated phases of gene repression remain to be explained. This unbiased approach toward expression characterization further implicated a number of previously unappreciated reg- ulatory molecules as playing a role in the development of the adipocyte phenotype in vitro. Regulation of RNA and protein levels of the known adipogenic transcription factors, e.g. C/EBP, C/EBP, C/EBP, PPAR, and SREBP-1, is not suffi- cient to generate the level of complexity seen during 3T3-L1 differentiation. For example, the kinetics of different known targets of these genes can in some cases be markedly different, e.g. PPAR targets aP2 and phosphoenolpyruvate carboxyki- nase. This observation suggests that many other genes either modulate the behavior of these known factors or act independ- ently to direct adipose gene expression. This study identifies a FIG.4. Comparison of preadipocyte and adipocyte gene ex- large number of such candidate regulatory molecules including pression levels in vivo and in vitro. 1435 genes that were enriched transcription factors, transcriptional coactivators, and signal- in preadipocytes or adipocytes were grouped by k-means clustering ing molecules. These data can now be analyzed further to test using a dot product metric according to their absolute level of expres- whether some of these factors can account for the regulation sion in 10 3T3-L1 time points and in isolated adipocytes and stromal cells from wild-type and ob/ob white adipose tissue. A, six clusters of evident in the clusters, the genes of which were expressed with genes labeled Adip1–Adip6 were enriched in adipocytes in vitro and in different kinetics from the clusters containing of PPAR, vivo and were expressed at quantitatively similar levels in those two C/EBP, SREBP-1, and their target genes. states. B, five clusters of genes labeled Preadip1–Preadip5 were more The relevance of these findings to adipogenesis in vivo was highly expressed in preadipocytes than cells that had accumulated cytoplasmic lipid both in vitro and in vivo. These clusters of genes evaluated further in a formal comparison of the expression provide novel markers for this unique population of preadipocyte cells. profile of in vitro preadipocytes and adipocytes to their in vivo C, four clusters of genes labeled Vivo1–Vivo4 were expressed at high counterparts. These data indicate that although adipocytes in levels in vivo and were undetectable or expressed at much lower levels vitro express many of the same genes as well as morphologic in vitro. One of these clusters, Vivo1, is enriched specifically in adipo- cytes in vivo and is lowly or not expressed in vitro. In cluster Vivo2, and metabolic characteristics of in vivo adipocytes, the expres- many genes were expressed in cells of the preadipocyte/adipocyte line- sion profile of preadipocytes and adipocytes in vitro are differ- age that were not expressed in vitro. In the clusters Vivo3 and Vivo4, ent from those of the stromal and adipocyte fractions of white genes were uniquely expressed in the stromal fraction isolated from adipose tissue in vivo. The key lipogenic enzymes ATP-citrate wild-type and ob/ob adipose tissue. These two clusters characterize the population of in vivo cells that includes preadipocytes and possibly lyase and phosphoenolpyruvate carboxykinase are expressed other cell types responsible for supporting the fully differentiated adi- at 20 –200-fold lower levels (at day 7) or 2–10-fold lower levels pocyte phenotype. Three clusters of genes labeled Vitro1–Vitro3 were (at day 28) in vitro relative to in vivo adipocytes, suggesting at more highly expressed in vitro and expressed at low or undetectable least one possible explanation for the lower total accumulation levels in vivo. of triglyceride in cultured adipocytes relative to in vivo adipo- scription factors C/EBP, PPAR2, SREBP-1, C/EBP, cytes. These results suggest that other heretofore unknown factors are necessary for the development of the fully differen- C/EBP, CHOP-10, AEBP1, COUP-TF (4, 26, 27), and others (see arrays.rockefeller.edu/obesity/adipocyte for the complete tiated adipocyte in vivo. The data also suggest that preadipo- cytes in vivo exhibit a novel phenotype that is not entirely list of differentially expressed molecules). A large group of genes was repressed during in vitro adipo- mimicked by preadipocytes in vitro. These cells express a num- genesis. One particular phase of interest is a large group of cell ber of genes with immune functions, suggesting an even cycle-related genes (Fig. 2R). 3T3-L1 cells, after the addition of broader array of roles for the in vivo preadipocyte than previ- growth factors at confluence, go through a clonal expansion ously appreciated. Global Expression Profiles of Adipogenesis 34173 TABLE I Genes responsive to adipocyte differentiation in vivo and in vitro Genes from adipocyte-specific (common to in vivo and in vitro), preadipocyte-specific (common to in vitro and in vivo), in vivo specific, and in vitro specific clusters are shown along with the cluster they were present in from Fig. 4. Transcription factors are shown in bold type. Complete membership of these clusters is available online at arrays.rockefeller.edu/obesity/adipocyte. In vivo and in vitro adipocyte In vivo and in vitro preadipocyte In vivo enriched genes In vitro enriched genes enriched genes enriched genes Gene Cluster Gene Cluster Gene Cluster Gene Cluster ACRP30/AdipoQ/Adiponectin Adip1 HMGl-Y Preadip1 ATP-Citrate Lyase Vivo1 PREF1 Vitro1 Adipsin Adip1 IFN Preadip1 Caveolin-1 Vivo1 Osteoblast Spec. 1 Vitro1 Aldehyde DH Adip1 Nuclear LIM Preadip1 PEPCK Vivo1 Mdm2 Vitro1 interactor Angiotensinogen Adip1 Prx2 Homeobox Preadip1 Acetyl-CoA Synthetase Vivo1 mrp/plf3 proliferin Vitro1 aP2 Adip1 TSC-36 Preadip1 Frizzled4 Vivo1 Annexin VIII Vitro1 Fat Specific Protein 27 Adip1 -Amylase Preadip2 Skeletal LIM FHL 1 Vivo1 Id HLH Factor Vitro1 Glycerophosphate DH Adip1 a-B2 Crystallin Preadip2 17-hydroxysteroid DH Vivo1 Apoptosis signal-reg Vitro2 kinase1 Haptoglobin Adip1 AEBP1 Preadip2 High MW GH Receptor Vivo1 Sox-4 Vitro2 Hormone Sensitive Lipase Adip1 Id related Preadip2 TSH Receptor Vivo1 AP-2 Vitro2 LAF1 Transketolase Adip1 junB Preadip2 obese mRNA Vivo1 FK506BP-13 Vitro2 Long Chain Fatty Acyl-CoA Adip1 Kruppel-like factor Preadip2 TNF Vivo2 Galactokinase Vitro2 Synthetase Transferrin Adip1 MCSF Preadip2 Zn Finger A20 Vivo2 p160 myb BP Vitro2 ACTH Receptor Adip2 PAI-1 Preadip2 SOCS-3 Vivo2 Proliferin Vitro2 Acyl-CoA BP Adip2 Spi2 Preadip2 C1q a-chain Vivo2 FK506BP-51 Vitro2 Angiotensinogen Adip2 FGF Receptor Preadip3 C1q b-chain Vivo2 HMGI-C Vitro2 ApoC1 Adip2 Forkhead Box F2 Preadip3 C1q c-chain Vivo2 cyclin G Vitro3 3 Adrenoceptor Adip2/3 Histone Deacetylase Preadip3 PDGF-inducible JE Vivo2 FK506BP-65 Vitro3 HD1 C/EBP Adip2 LCAT Preadip3 PDGF-inducible KC Vivo2 Zn Finger HRX/ Vitro3 ALL-1 CPT II Adip2 Transcription Preadip3 MIP-2 Vivo2 Keratinocyte Growth Vitro3 Factor E2a Factor Citrate Transporter Adip2/3 Zn Finger Zic3 Preadip3 IL-1b Vivo2 Frizzled Vitro3 GLUT-4 Adip2 c-myc Preadip4 IL-6 Vivo2 SOCS-2 Vitro3 Insulin Activated AA Adip2 C/EBP Preadip4 c-fos Vivo2 Pbx3b Vitro3 Transporter Malate DH Adip2 Endothelial MAP1 Preadip4 fosE Vivo2 C/EBP Vitro3 Malic Enzyme Adip2 TNF-Receptor-1 Preadip4 IP-10 Cytokine Vivo2 MEST Adip2 Erythroid Kruppel- Preadip4 Zn Finger p36 Vivo2 like Monoglyceride Lipase Adip2 204 interferon Preadip5 zif/268/Krox24 Vivo2 activatable p18 Adip2 ApoD Preadip5 LRG-21 Vivo2 p19 Adip2 fos-related antigen Preadip5 C10-like Chemokine Vivo2 PPAR2 Adip2 HES-1 HLH Preadip5 Gut Enriched Kruppel- Vivo2 like Spot14 Adip2 Mbh-1 Preadip5 Krox-20 Vivo2 SREBP-1 Adip2 Mkr3 Zn Finger Preadip5 ICAM-1 Vivo2 Glucose-6-P DH Adip3 OSF-2 Preadip5 RDC1 Chemokine Receptor Vivo3/4 Histone H1 Adip3 p85 Secreted Preadip5 junB Vivo3 TOR/ROR Adip3 PDGF Receptor Preadip5 Enkephalin Vivo3 VLDL Receptor Adip3 Prostacyclin Preadip5 Cathepsin S Vivo3 Synthase FPP Synthetase Adip3 EF1 Preadip5 Cathepsin H Vivo3 3-Phosphogylcerate DH Adip4 UCP-2 Preadip5 CXCR-4 Vivo3 Fatty Acid Synthase Adip4 VCAM-1 Preadip5 IL-1 Vivo3 Low MW GH Receptor Adip4 PAI-2 Vivo3 PFK-1 Adip4 PU.1 Vivo3 Mal1 Lipid Binding Adip4 C10 Vivo3 Pyruvate Carboxylase Adip4 Cor1 Basic HLH Factor Vivo4 RXRa Adip4 Cytokine Receptor-like EBI3 Vivo4 SCD1 Adip4 Prostaglandin E Receptor Vivo4 Transaldolase Adip4 PECAM-1 Vivo4 Triosephosphate Isomerase Adip4 EBI-1 G-protein Receptor Vivo4 VEGF Adip4 TRAF-1 Vivo4 VEGF-b Adip4 IL-3 Receptor Vivo4 SCD2 Adip5 Angiogenin Vivo4 Lactate DH Adip5 Endothelin B Receptor Vivo4 Enolase Adip5/6 Macrophage Specific NRAMP Vivo4 PHAS II Adip5 Macrophage Metalloelastase Vivo4 Cyclin D Adip5 MIP-1 Vivo4 Cyclin E Adip5 Neutrophil Cytosol Factor2 Vivo4 3-hydroxyacyl-CoA DH Adip6 IL-4 Receptor Vivo4 Adipocyte p27 Adip6 IL-10 Vivo4 CHOP-10 Adip6 RANTES Vivo4 Cytosolic Malate DH Adip6 Lymphoid Spec. IFN Reg. Vivo4 Fact Dihydrolipoamide DH Adip6 Nurr1 Nuclear Orphan Vivo4 Receptor p21/Waf1 Adip6 Prostaglandin Synthase Vivo4 Aldolase A Adip6 34174 Global Expression Profiles of Adipogenesis 1108 –1112 These in vivo expression data provide a new framework in 12. Tanaka, T., Yoshida, N., Kishimoto, T., and Akira, S. (1997) EMBO J. 16, which the functional significance of observed changes in vari- 7432–7443 13. Rosen, E. D., Sarraf, P., Troy, A. E., Bradwin, G., Moore, K., Milstone, D. S., ous in vitro models of adipogenesis can be evaluated. Finally, Spiegelman, B. M., and Mortensen, R. M. (1999) Mol. Cell 4, 611– 617 these data suggest the importance of evaluating cell culture 14. Barak, Y., Nelson, M. C., Ong, E. S., Jones, Y. Z., Ruiz-Lozano, P., Chien, K. R., models for studying in vivo phenomena using microarrays. Koder, A., and Evans, R. M. (1999) Mol. Cell 4, 585–595 15. 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Published: Sep 1, 2001

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