Research Article 889

PKB␣ is required for adipose differentiation of mouse embryonic fibroblasts

Anne Baudry, Zhong-Zhou Yang and Brian A. Hemmings* Friedrich Miescher Institute for Biomedical Research, Maulbeerstr. 66, CH-4058, Basel, Switzerland *Author for correspondence (e-mail: [email protected])

Accepted 14 November 2005 Journal of Cell Science 119, 889-897 Published by The Company of Biologists 2006 doi:10.1242/jcs.02792

Summary kinase B␣ (PKB␣) is a key regulator of metabolism, downregulated in PKB␣-deficient MEFs but could be proliferation and differentiation. We have explored the role restored by expressing an active PKB␣ in the deficient cells. of PKB␣ in adipogenesis using wild-type and PKB␣- The level of lipocalin 2, renin 1 and receptor-activity- knockout mouse embryonic fibroblasts (MEFs) and show modifying protein 3 expressed by adipose cells was that lack of PKB␣ prevents MEF differentiation into also decreased in PKB␣-deficient MEFs, and are inhibited adipocytes. Expression of ectopic PKB␣ in PKB␣-deficient by LY294002 treatment during early adipocyte cells restores adipogenesis. We identified 80 genes whose differentiation of 3T3-L1 cells. The results underscore an expression was upregulated in wild-type MEFs during essential role for PKB␣ in the transcriptional program adipogenesis but whose expression was significantly required for adipogenesis. reduced in PKB␣-deficient MEFs under the same conditions. Significantly, the regulator of adipogenesis Key words: PKB␣, Adipocyte differentiation, Mouse embryonic Krüppel-like transcription factor 15 expression was fibroblasts, Microarray analysis

Introduction many cellular processes stimulated by and growth Adipogenesis is a complex process with multiple steps factors (Brazil and Hemmings, 2001; Lawlor and Alessi, 2001; including growth arrest, clonal expansion, withdrawal from the Whiteman et al., 2002). The three members of the PKB family, cell cycle and terminal differentiation (Gregoire et al., 1998; PKB␣ (Akt1), PKB␤ (Akt2), and PKB␥ (Akt3), are encoded Rosen et al., 2000). In parallel, adipose conversion is by distinct genes but share a similar structural organisation

Journal of Cell Science accompanied by temporally regulated expression of numerous (Datta et al., 1999; Scheid and Woodgett, 2001; Hill and genes. Some of these genes encode transcription factors, such Hemmings, 2002). It remains a matter of debate whether these as members of the CCAAT/enhancer binding protein (C/EBP) isoforms have specific or redundant functions in vivo. This family and peroxisome proliferator-activated receptor ␥ issue was addressed recently by disruption of different PKB (PPAR␥). These are key regulators of this transcriptional genes in mice (Yang et al., 2004). PKB␤-deficient mice exhibit program and promote the expression of most genes and a diabetes-like syndrome (Cho et al., characterising fat cells, such as the adipocyte-specific fatty- 2001a; Garofalo et al., 2003). Depending on the genetic acid-binding protein aP2 (Spiegelman et al., 1993). background, PKB␤-knockout mice are also characterised by Evidence has accumulated that insulin and insulin-like moderate growth deficiency as well as age-dependent decrease -1 (IGF-1) signalling pathways play a significant in mass (Garofalo et al., 2003). However, MEFs role in adipogenesis. Both are essential inducers of adipocyte derived from these mutants are not significantly different to differentiation (Smith et al., 1988). Insulin receptor-deficient wild-type MEFs in their ability to differentiate into adipocytes mice display underdeveloped adipose tissue (Cinti et al., 1998) (Peng et al., 2003). Unlike PKB␤-deficient mice, PKB␣- and mouse embryonic fibroblasts (MEFs) derived from these knockout mice have normal glucose homeostasis but display mutants fail to differentiate into adipocytes (Nakae et al., deficient placental development, which results in growth 2003). Furthermore, a double-knockout deficiency of insulin retardation and enhanced neonatal morbidity (Chen et al., receptor substrate-1 (IRS-1) and IRS-2 results in failure of the 2001; Cho et al., 2001b; Yang et al., 2003). Subcutaneous fat adipocyte developmental program (Miki et al., 2001). Finally, is also moderately reduced in the absence of PKB␣, phosphatidylinositol 3-kinase (PI 3-kinase) activity transiently implicating this isoform in adipogenesis (Yang et al., 2003). increases during conversion of preadipocytes into adipocytes Finally, the major characteristic of PKB␥-knockout mice is (Sakaue et al., 1998), and the presence of PI 3-kinase inhibitors reduction in brain size (Easton et al., 2005; Tschopp et al., completely blocks the differentiation process (Tomiyama et al., 2005). The phenotypic differences between these PKB- 1995; Christoffersen et al., 1998; Xia and Serrero, 1999), knockout mice suggest that PKB isoforms participate jointly in indicating that the PI 3-kinase pathway is also involved in the regulation of some cellular processes, such as growth, but adipogenesis. also exert specific roles in vivo, for example in glucose The protein kinase B (PKB, also named Akt) is a metabolism. downstream target of PI 3-kinase and plays a critical role in Previous studies have suggested a role for PKB in 890 Journal of Cell Science 119 (5)

adipogenesis, because expression of a constitutively activated form of PKB in 3T3-L1 pre-adipose cells caused spontaneous differentiation into adipocytes (Kohn et al., 1996; Magun et al., 1996). In the current study, we investigated the role of the ␣ isoform of PKB in adipose differentiation. We show that MEFs derived from PKB␣-deficient mice fail to differentiate into adipocytes in vitro and identified target genes regulated by PKB␣ during the early steps of adipocyte conversion.

Results Defective adipogenesis in the absence of PKB␣ To investigate the role of PKB␣ in adipocyte differentiation, MEFs were derived from wild-type and PKB␣–/– embryos from the same litter and thus have an identical genetic background. They were induced to differentiate into adipocytes in vitro using a standard adipogenic induction cocktail of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), the synthetic glucocorticoid dexamethasone and insulin, followed by addition of insulin every 2 days (see Materials and Methods for details). After 8 days of differentiation, Oil Red Fig. 1. Inhibition of adipogenesis in PKB␣–/– MEF cells. (A) Two O staining was performed to monitor intracellular lipid days after confluence, MEF cells were induced to differentiate into accumulation. Wild-type MEFs cultured in the presence of adipocytes with IBMX, dexamethasone and insulin for 48 hours differentiating agents accumulated fat droplets but no obvious followed by insulin alone every 2 days. Cells were stained with Oil- lipid accumulation was observed in PKB␣–/– MEFs (Fig. 1A). Red O at 8 days post-induction. (B) Expression of adipocyte-specific ␣–/– genes after stimulation of the adipose differentiation process in wild- This failure to support lipid storage in PKB MEFs persisted ␣–/– for at least 16 days following induction of differentiation (data type and PKB MEFs. Total RNA was extracted from cells at the times indicated and analysed by RT-PCR as described in the not shown). As the adipogenic process is characterised by Materials and Methods. expression of specific adipocyte markers such as PPAR␥ and aP2, we analysed expression of these two transcripts in both cell types by semi-quantitative RT-PCR. Whereas treatment of wild-type MEFs with adipogenic inducers led to the induction of Pparg and aP2 mRNA, no change in mRNA levels for either marker was detected in the absence of PKB␣ (Fig. 1B). The three PKB isoforms (␣, ␤, ␥) are expressed in wild-type

Journal of Cell Science MEFs (Peng et al., 2003). Western blot analysis using isoform- specific antibodies was performed to determine the expression profile of the three isoforms during differentiation in wild-type and PKB␣–/– MEFs. The data presented in Fig. 2A confirm the absence of PKB␣ protein in PKB␣–/– MEFs and also showed that the amounts of PKB␤ and ␥ were not significantly different in PKB␣–/– and wild-type MEFs, precluding any compensatory upregulation of PKB␤ and/or ␥ in the absence of the ␣ isoform. In addition, the activation status of PKB was monitored during adipose conversion using phospho-specific PKB antibodies that recognise the two crucial phosphorylation sites of PKB present in all three isoforms: Thr308 (in PKB␣) in the activation loop of the kinase domain and Ser473 (in PKB␣) in the hydrophobic motif (Alessi et al., 1996; Meier et al., 1997; Brodbeck et al., 1999). PKB activity increased during differentiation of wild-type MEFs whereas this activity is reduced in the absence of PKB␣ expression (Fig. 2B). Taken Fig. 2. Levels of the three PKB isoforms and the phosphorylation together, these results show that the PKB␣ isoform is necessary status of PKB in wild-type and PKB␣–/– MEFs during adipose for the adipogenic differentiation of MEFs. conversion. Total cellular extracts were prepared from cells before (day 0) or at 2, 5, 8 and/or 12 days after adipose induction treatment ␣ ␤ ␥ Ectopic expression of PKB␣ restores adipogenesis in and analysed by western blotting with PKB -, PKB - and PKB - ␣–/– specific antibodies (A) or with phospho-PKB specific antibodies PKB MEFs (pSer473 and pThr308) (B). Blots were reprobed with anti-actin To examine whether the impairment of adipocyte antibodies to control for equal loading. differentiation observed in PKB␣–/– MEFs is specifically due to the absence of PKB␣, the cells were infected with a retrovirus expressing HA-PKB␣ (Fig. 3A). Ectopic expression intracellular lipid accumulation after 15 days of differentiation of PKB␣ in PKB␣–/– MEFs resulted in a marked increase in as shown by Oil Red O staining (Fig. 3B). Moreover, PKB␣–/– PKB␣ and adipogenesis 891

Fig. 3. Ectopic expression of PKB␣ by retrovirus infection restores adipogenesis in PKB␣–/– MEFs. (A) PKB␣–/– MEFs were infected with retrovirus expressing HA-PKB␣ or vector only. After puromycin selection, extracts of cells were analysed by western blotting for the expression of HA-PKB␣. (B) Cells were exposed to the adipogenic cocktail and stained with Oil Red O after 15 days. (C) RT-PCR analysis of adipose markers PPAR␥ and aP2 in PKB␣–/– MEFs infected with vector or HA-PKB␣-expressing retrovirus and submitted to adipose differentiation.

respond in the absence of PKB␣. The genes were classified into ten clusters based upon the temporal expression profiles of adipogenic induction of wild-type MEFs (Table 1). Of these, 24 genes were strongly upregulated after 2 hours and then declined rapidly (clusters 1, 2) or declined slowly (cluster 3) to the basal levels, or showed a plateau at 6 hours before reduction of expression (cluster 4) (Fig. 4). Twenty-three genes increased after 6 hours and then declined to basal values (cluster 5) or maintained a moderate or high level of expression (clusters 6, 7) (Fig. 4). Clusters 8 and 9 comprise genes whose MEFs expressing a constitutively active form of PKB␣ (m/p- expression progressively increased in wild-type MEFs HA-PKB␣) were able to spontaneously differentiate into following induction of differentiation. Expression profiles of adipocytes in the absence of adipogenic treatment (data not cluster 8 were not modified in the absence of PKB␣, whereas shown). Finally, retroviral introduction of PKB␣ promoted expression of genes in cluster 9 was enhanced in PKB␣–/– Pparg and aP2 during the adipose conversion MEFs after 24 hours of adipogenic treatment and then declined process (Fig. 3C). to basal values (Fig. 4). Cluster 10 includes all otherwise unclassified genes. To validate the microarray results, the Gene expression profiles after adipogenic treatment of expression profiles of some genes with a marked difference wild-type and PKB␣–/– MEFs between wild-type and PKB␣–/– MEFs were determined by To identify genes regulated by PKB␣ during the early stages semi-quantitative RT-PCR (data not shown). The results of adipogenesis, total RNA was extracted from wild-type and obtained by this method were similar to the microarrays. ␣–/– Journal of Cell Science PKB MEFs at 2 days postconfluence (time 0) and 2, 6, 24, 48 hours after induction of differentiation. Transcript levels PKB␣ is required for Krüppel-like factor 15 (Klf15) were determined by microarray with MOE430A Affymetrix expression during adipose differentiation of MEFs gene chips, which detect 22,690 distinct mouse genes and In the microarray experiment, the gene encoding transcription ESTs. Adipogenesis is characterised by the expression of key factor KLF15 was induced by the adipogenic cocktail in wild- regulator genes, such as members of the C/EBP family. Yeh type MEFs but was only basally expressed in PKB␣–/– MEFs and collaborators found that mRNA levels of the transcription (Fig. 5A). It was recently demonstrated that KLF15 increases factors C/EBP␦ and C/EBP␤ were transiently induced during during adipose conversion of 3T3-L1 cells and plays an adipose conversion of 3T3-L1 cells (Yeh et al., 1995). In our important role in this process (Gray et al., 2002; Mori et al., microarray experiment, we also observed a transient increase 2005). We further analysed the expression pattern of Klf15 in in mRNA levels for both C/EBP␦ and C/EBP␤ 2 hours after wild-type and PKB␣–/– MEFs by semi-quantitative RT-PCR adipogenic treatment of wild-type MEFs, validating the after 0, 1, 2, 5 and 8 days of adipose differentiation. As microarray results (data not shown). Similar induction of these expected, treatment of wild-type MEFs with the adipogenic two genes was also observed with PKB␣–/– MEFs, indicating cocktail resulted in the rapid induction and maintenance of that PKB␣ is not necessary for the expression of C/EBP␦ and Klf15 mRNA throughout the 8 days of differentiation. In C/EBP␤ (data not shown). To identify genes whose expression contrast with PKB␣–/– MEFs, this transcription factor is only during the first steps of adipocyte differentiation is PKB␣- modestly expressed with a transient and delayed induction after dependent, we first selected genes that displayed an at least 2 days which could be due to the other PKB isoforms on Klf15 twofold change relative to time 0 of wild-type MEFs in at least expression (Fig. 5B). We also observed that after 24 hours of one of the time points and a significant difference (P<0.05) in adipogenic treatment, retroviral-mediated PKB␣ expression in a one-way ANOVA. Only genes expressed at similar levels at PKB␣–/– MEFs reversed decreased expression of Klf15 (Fig. time 0 in wild-type and PKB␣–/– MEFs were further analysed. 5C). To further investigate regulation of Klf15 by PKB␣, we Of the 1103 genes satisfying these criteria, only those showing assessed Klf15 expression in PKB␣–/– MEFs expressing a at least a twofold difference in expression between wild-type constitutively active form of PKB␣ (Fig. 5D). In these cells, and PKB␣–/– MEFs in at least one time point were selected. Klf15 mRNA was markedly induced 3 days postconfluence This yielded 80 genes that were significantly upregulated compared with control vector-infected cells (Fig. 5E). The during the differentiation of wild-type MEFs but did not increase in Klf15 gene expression was accompanied by an 892 Journal of Cell Science 119 (5)

Table 1. Genes upregulated during the early initiation of adipose conversion in wild-type MEFs are fewer or not expressed at all in the absence of PKB␣* Affymetrix GenBank Cluster identification Symbol accession number Gene name Functional category Cluster 1 1420590_at Has1 NM_008215 Hyaluronan synthase 1 Signalling 1421240_at Ern1 NM_023913 Endoplasmic reticulum to nucleus signalling 1 Stress response 1418052_at Mvk BC005606 Mevalonate kinase Metabolism 1448862_at Icam2 NM_010494 Intercellular adhesion molecule 2 Cluster 2 1419431_at Ereg NM_007950 Epiregulin Growth factor 1421134_at Areg NM_009704 Amphiregulin Growth factor 1433494_at Dos BB180184 Downstream of Stk11 – ESTs : BB049138, BC016562 – Cluster 3 1416308_at Ugdh NM_009466 UDP-glucose dehydrogenase Transport 1418295_s_at Dgat1 BC003717 Diacylglycerol O-acyltransferase 1 Metabolism 1424296_at Gclc BC019374 Glutamate-cysteine ligase, catalytic subunit Glutathione biosynthesis 1424735_at Slc25a25 BC019978 Solute carrier family 25 Transport 1448666_s_at Tob2 AV174616 Transducer of ERBB2 – 1452257_at Bdh BF322712 3-Hydroxybutyrate dehydrogenase Metabolism 1455002_at Ptp4a1 AV331223 Protein tyrosine phosphatase 4a1 Signalling EST: BB817847 – Cluster 4 1420401_a_at Ramp3 NM_019511 Receptor activity modifying protein 3 Signalling 1417268_at Cd14 NM_009841 CD14 antigen Immune response 1424457_at Apbb3 BC024809 Amyloid beta precursor protein binding 1448029_at Tbx3 AA543734 T-box 3 protein Transcription 1449201_at Star L36062 Steroidogenic acute regulatory protein Steroid biosynthesis 1430164_a_at Grb10 AK012646 Growth factor receptor bound protein 10 Signalling 1426663_s_at Pcanap6 BC024519 Prostate cancer associated protein 6 Transport Cluster 5 1422454_at Krt1-13 NM_010662 Keratin complex 1 organization 1422790_at Nppc NM_010933 Natriuretic peptide precursor type C cGMP biosynthesis Cluster 6 1418946_at St3gal1 NM_009177 ST3 beta-galactoside ␣2,3 sialyltransferase1 Glycosylation 1419100_at Serpina3n NM_009252 Serine proteinase inhibitor Endopeptidase inhibitor 1436566_at Rab40b AV364488 Rab40b, member RAS oncogene family Signalling 1436737_a_at Sorbs1 BB737680 Sorbin and SH3 domain containing protein Transport Cluster 7 1418937_at Dio2 AF177196 Deiodinase type II Hormone catabolism 1416927_at Trp53inp1 AW495711 Transformation related protein 53 Apoptosis 1420900_a_at Rab18 AW542340 RAB18, member Ras oncogene family Signalling 1418601_at Aldh1a7 NM_011921 Aldehyde dehydrogenase family 1 Metabolism

Journal of Cell Science 1418677_at Actn3 NM_013456 Actinin alpha 3 Muscle contraction 1421921_at Serpina3m BC011158 Serine proteinase inhibitor Endopeptidase inhibitor 1425985_s_at Masp1 AB049755 Mannan-binding lectin serine protease 1 Proteolysis

Table continued on next page.

augmentation of Pparg expression (Fig. 5E). These data thus reduced the mRNA levels of these three genes (Fig. 6D), as suggest that PKB␣ is involved in the regulation of Klf15, itself well as the mRNA level of Klf15 (Fig. 6E). Taken together, the a key regulator in the transcription factor network controlling results indicate that the expression of Lcn2, Ramp3, Ren1 and adipogenesis. Klf15 is dependent on PI 3-kinase and PKB and these genes are good candidates to be involved in the early stages of Decreased expression levels of Lcn2, Ramp3, Ren1 and adipogenesis. Klf15 by inhibition of PI 3-kinase in 3T3-L1 cells Of the 80 genes identified by microarray analysis, several Discussion genes, such as lipocalin 2 (Lcn2), receptor-activity-modifying In this study, we have demonstrated that the ␣ isoform of PKB protein 3 (Ramp3) and renin 1 (Ren1), were robustly expressed plays a crucial role in the process of MEF differentiation into in wild-type MEFs after adipogenic treatment but did not adipocytes. We have shown that adipose conversion is impaired respond in the mutant MEFs (Fig. 6A,B). Using semi- in the absence of PKB␣, whereas ectopic expression of PKB␣ quantitative RT-PCR, we further studied the expression profile in PKB␣-deficient cells leads to accumulation of lipid droplets of these genes during the first steps of adipocyte differentiation and expression of adipocyte-specific genes, such as Pparg and of 3T3-L1 cells, which is the most widely used model for aP2. We did not observe any upregulation of PKB␤ and PKB␥ investigating mechanisms of adipogenesis in vitro (Green and compensating for the absence of PKB␣ in PKB␣–/– MEFs. Meuth, 1974). Transcript levels of Lcn2, Ramp3 and Ren1 Thus, PKB␣ is the major isoform of PKB involved in adipocyte increased after a few hours in the presence of the adipogenic differentiation of MEFs. cocktail, with a pattern similar to that observed in wild-type Furthermore, we have identified several genes whose MEFs (Fig. 6D). Addition of LY294002, an inhibitor of PI 3- expression is significantly upregulated during the early steps of kinase that leads to a decrease in PKB activity (Fig. 6C), adipocyte differentiation in wild-type MEFs compared with PKB␣ and adipogenesis 893

Table 1. Continued Affymetrix GenBank Cluster identification Symbol accession number Gene name Functional category Cluster 7 1427056_at Adamts15 AV228731 A disintegrin-like and metalloprotease with Proteolysis thrombospondin type 1 motif 1448181_at Klf15 BC013486 Kruppel-like factor 15 Transcription 1427747_a_at Lcn2 X14607 Lipocalin2 Transport 1448975_s_at Ren1 NM_031192 Renin 1 Proteolysis 1450890_a_at Abi1 AW912678 Abl-interactor 1 Cell growth 1452579_at Nifun AK009021 NifU-like domain containing protein – EST: BC025600, BE981853, BQ175154, AV012400 Cluster 8 1416918_at Dlgh3 NM_016747 Discs, large homolog 3 Protein binding 1417851_at Cxcl13 AF030636 Chemokine (C-X-C motif) ligand 13 Immune response 1421114_a_at Dspg3 NM_007884 Dermatan sulphate proteoglycan 3 – 1418858_at Aox3 NM_023617 Aldehyde oxidase 3 Transport 1422905_s_at Fmo2 NM_018881 Flavin containing monooxygenase 2 Transport 1423436_at Gsta3 AI172943 Glutathione S-transferase – 1424921_at Bst2 BC008532 Bone marrow stromal cell antigen 2 – 1427944_at C1qdc1 BE951890 C1q domain 1 – 1451038_at Apln BB819942 Apelin Signalling 1451932_a_at Tsrc1 BC016215 Thrombospondin repeat protein 1 – 1433855_at Abat BF462185 4-Aminobutyrate aminotransferase Catabolism Cluster 9 1422948_s_at Hist1h4 NM_013550 Histone 1, H4j, b, h, k, m, i, c, a Nucleosome organisation 1424854_at Hist1h4 BC019757 Histone 1, H4f, b, m, i, a Nucleosome organisation Cluster 10 1451558_at Fbxw7 AV338062 F-box and WD-40 domain protein 7 Signaling 1427893_a_at Pmvk BI713896 Phosphomevalonate kinase Metabolism 1449108_at Fdx1 D43690 Ferredoxin 1 Transport 1452160_at Tiparp BB707122 TCDD-inducible poly(ADPribose) polymerase Ribosylation 1417374_at Tuba4 AW491660 Tubulin, alpha 4 Structure 1439083_at Ahi1 BM239359 Abelson helper integration site – 1424304_at Tpcn2 BC025890 Two pore segment channel 2 Transport 1418865_at Zfp385 NM_013866 Zinc finger protein 385 RNA binding 1418854_at Birc2 NM_007465 Baculoviral IAP repeat protein 2 Apoptosis 1416034_at Cd24a NM_009846 CD24a antigen Immune response 1448499_a_at Ephx2 NM_007940 Epoxide hydrolase 2, cytoplasmic Metabolism 1449525_at Fmo3 NM_008030 Flavin containing monooxygenase 3 Transport 1423915_at Olfml2b BC025654 Olfactomedin-like 2B – 1427345_a_at Sult1a1 AK002700 Sulfotransferase Metabolism 1420928_at St6gal1 BG075800 Beta galactoside alpha 2,6 sialyltransferase 1 Glycosylation

Journal of Cell Science 1435906_x_at Gbp2 BE197524 Guanylate nucleotide binding protein 2 Immune response 1416530_a_at Pnp BC003788 Purine-nucleoside phosphorylase – EST: AK019474, BG074158, BM248225 –

*The microarray data have been deposited at The NCBI gene expression omnibus (GEO) with accession number GSE2746.

PKB␣–/– MEFs. These include the transcription factor KLF15, express high levels of PPAR␥. The PKB␣/PKB␤-deficient which was recently identified as an essential regulator of MEFs partially recover adipocyte differentiation after infection adipogenesis. We have shown that PKB␣–/– MEFs with retrovirus expressing PPAR␥ (Peng et al., 2003). Here, we overexpressing a wild-type PKB␣ or a constitutively active have demonstrated that MEFs devoid of the PKB␣ isoform membrane-targeted PKB␣ exhibit increased levels of Klf15. display defective adipocyte differentiation and that Changes in expression of genes such as Lcn2, Ramp3 and Ren1 reintroduction of PKB␣ into these cells can restore lipid have been demonstrated in wild-type MEFs as well as 3T3-L1 accumulation and expression of Pparg and aP2. Interestingly, cells after the initiation of adipocyte conversion. In addition, the ability of PKB␤–/– MEFs to differentiate into adipocytes is inhibition of PKB activity in the presence of LY294002 leads not significantly different to that of wild-type MEFs (Peng et to decreased levels of these three genes as well as Klf15 in 3T3- al., 2003). Thus, it is likely that PKB␣ is the major PKB L1 cells. These data suggest the involvement of the PI 3- isoform involved in adipogenesis in MEFs. Using small kinase/PKB pathway in the regulation of Klf15, Lcn2, Ramp3 double-strand RNA interference, Xu and Liao have also and Ren1 expression during the early phase of adipocyte observed that a decrease in PKB␣ in 3T3-L1 cells prevents differentiation. adipocyte differentiation (Xu and Liao, 2004). This indicates It was reported recently that mice deficient for both PKB␣ that PKB␣ involvement in adipogenesis is not restricted to our and PKB␤ display dwarfism and die immediately after birth cell models. (Peng et al., 2003). The mutant mice exhibit severe The Krüppel-like family of transcription factors play developmental defects in adipose tissue as well as in bone, skin important regulatory roles in cellular proliferation, development and muscle. Moreover, MEFs derived from these double- and differentiation. We have demonstrated that Klf15 is knockout mice are unable to differentiate into adipocytes or to upregulated during the first steps of adipocyte differentiation of 894 Journal of Cell Science 119 (5)

5 Cluster 1 6 Cluster 4 4 Cluster 7 5 4 3 3 4 3 2 2 2 1 1 1 0 0 0 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48

25 Cluster 2 8 Cluster 5 5 Cluster 8 20 6 4 15 3 Fig. 4. Cluster map of 4 genes upregulated during 10 2 an early step of adipose 5 2 1 differentiation in wild-type MEFs showing little or no 0 0 0 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48 upregulation in PKB␣–/– MEFs. The genes identified by microarray 6 Cluster 3 5 Cluster 6 4 Cluster 9 analysis were grouped into 5 Relative expression (fold induction) 4 3 ten clusters according to 4 3 their temporal profile of 3 2 expression. Values are the 2 2 average fold induction 1 1 after adipogenic treatment 1 of all genes in each cluster. 0 0 0 Cluster 10 which 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48 comprises all otherwise Time (h) Time (h) Time (h) unclassified genes is presented in Table 1. Wild-type MEFs PKBα-/- MEFs Journal of Cell Science

Fig. 5. PKB␣ induces Klf15 during adipose conversion of MEFs. (A) Microarray profile of Klf15 in wild-type and PKB␣–/– MEFs during the first 2 days after hormonal stimulation. (B) Total RNA was extracted from cells at the times indicated and analysed by RT-PCR. (C) RT-PCR analysis of Klf15 gene expression after 24 hours of adipogenic treatment of PKB␣–/– MEFs infected with vector or HA-PKB␣ expressing retrovirus. (D) PKB␣–/– MEFs were infected with retrovirus expressing (m/p)-HA-PKB␣ or vector only. Following puromycin selection, lysates were analysed by western blotting for the expression of a constitutively active form of PKB␣. (E) Total RNA was extracted from cells 3 days postconfluence and analysed by RT-PCR as described in the Materials and Methods. PKB␣ and adipogenesis 895

Fig. 6. Inhibition of the PI 3-kinase/PKB signalling pathway reduces expression of Lcn2, Ramp3, Ren1 and Klf15 during the first stages of adipose differentiation of 3T3-L1 cells. Microarray profiles (A) and RT-PCR analysis (B) of Lcn2, Ramp3 and Ren1 genes in wild-type and PKB␣–/– MEFs during the first 2 days of adipose treatment. (C,D) 3T3-L1 cells were starved for 24 hours and then treated with LY294002 or not (DMSO only) 20 minutes before and every 12 hours after addition of the adipose cocktail. Lysates and RNA from cells were collected at the times indicated. Total cellular were subjected to western blot analysis with phospho-PKB-specific antibodies (pSer473) and actin as a loading control (C) and total RNA was submitted to RT-PCR analysis for Ramp3, Lcn2, Ren1 (D) or Klf15 (E). Journal of Cell Science

wild-type MEFs but not in the absence of PKB␣. Several of adipogenesis. The single transmembrane domain protein studies have shown recently that members of the KLF family Ramp3, which is expressed in rat fat tissue (Nagae et al., 2000), are essential for adipogenesis. KLF2 decreases rapidly upon is an accessory protein for the G-protein-coupled receptor adipocyte differentiation of 3T3-L1 cells and has been (GPCR) and is involved in trafficking and folding (Morfis et described as a negative regulator of adipogenesis through al., 2003). Several studies have shown that Ramp3, associated inhibition of Pparg (Banerjee et al., 2003). By contrast, KLF5 with calcitonin-receptor-like receptor, can act as a receptor for was shown to be required for adipocyte conversion of 3T3-L1 adrenomedullin, but it appears that Ramp3 may also regulate cells (Oishi et al., 2005). KLF15 is highly expressed in adipose other receptors. Interestingly, it was reported recently that tissue and also during differentiation of 3T3-L1 cells into expression and secretion of adrenomedullin are more obvious adipocytes (Gray et al., 2002). It was demonstrated recently by in 3T3-L1 preadipocytes than in adipocytes (Li et al., 2003). RNA interference and expression of a dominant-negative These observations are consistent with evidence presented here mutant of KLF15 that this transcription factor acts in that Ramp3 is a good candidate to be involved in the first hours adipogenesis through the regulation of Pparg gene expression of adipocyte differentiation. (Mori et al., 2005). In this report, we have also shown that Klf15, Renin is the rate-limiting protease of the renin-angiotensin as well as Pparg, are enhanced in the absence of hormonal system (RAS) involved in the production of angiotensin II. The stimulation in PKB␣–/– MEFs overexpressing active PKB␣, RAS is widely known for its essential role in blood pressure which can spontaneously differentiate into adipocytes. It was regulation. It has been reported recently that all the main shown previously that PKB affects Pparg gene expression, at components of this system are also expressed in adipose tissue least in part, through the Forkhead transcription factor FOXO1 (Engeli et al., 2000; Ailhaud et al., 2002) but the involvement (Peng et al., 2003). Our findings now suggest that the effect of of the RAS in adipogenesis is not yet clearly established. PKB␣ on Pparg expression is also achieved through KLF15. Angiotensin II may be a potent inhibitor of adipose conversion Further work is now required to determine the mechanism of (Schling and Loffler, 2001) or it may indirectly stimulate how PKB␣ can upregulate Klf15 gene expression. adipocyte differentiation (Saint-Marc et al., 2001). In this The results presented here also show a strong and transient study, we found that induction of adipose conversion rapidly induction of Ramp3 transcripts within 48 hours after induction leads to an increase in Ren1 gene transcript in both wild-type 896 Journal of Cell Science 119 (5)

MEFs and 3T3-L1 cells, whereas Ren1 mRNA is almost were selected with 3-5 ␮g/ml puromycin (Sigma) for 6-8 days and resistant clones undetectable in PKB␣–/– MEFs or in the presence of an were expanded. inhibitor of PI 3-kinase. These results suggest that the PI 3- Microarray analysis kinase/PKB pathway regulates Ren1 expression and Total RNA was extracted from cells using TRIzol (Invitrogen) according to the underscore a possible role for the RAS in the process of manufacturer’s instructions. RNA was quantified by spectrometry and the quality confirmed by gel electrophoresis. cDNA was synthesised from 10 ␮g total RNA adipogenesis. using the SuperScript cDNA system (Invitrogen) and used to generate biotin- Lcn2, a member of the lipocalin family of small secreted labelled cRNA with the Enzo BioArray High Yield RNA transcript labelling kit proteins, has also been shown in this study to be strongly (Enzo Diagnostics, USA). After fragmentation, 10 ␮g cRNA was hybridised with the mouse MOE430A GeneChipsTM (Affymetrix, Santa Clara, USA) following the upregulated during the early stages of adipose differentiation. protocol recommended by Affymetrix. Genechips were then scanned in an Lipocalins bind small hydrophobic molecules and specific cell Affymetrix 2500 scanner and gene expression analysed using the GeneSpring surface receptors and form macromolecular complexes Software 7 (Silicon Genetics). Only genes with an expression value greater than (Flower, 1996). Interestingly, it was shown by a proteomic 100 in at least one condition were further examined. Genes were then selected for an at least twofold change relative to the control condition (time 0 of wild-type approach that the Lcn2 protein is secreted by 3T3-L1 MEFs) in at least one condition and the data submitted to one-way ANOVA (A value adipocytes (Kratchmarovat et al., 2002). Furthermore, Lcn2 of P<0.05 was considered significant). The resulting gene data were finally gene expression was found by microarray analysis to increase subjected to a Benjamini and Hochberg false-discovery rate multiple-testing more than fivefold at 24 hours, 4 and 7 days after initiation of correction and a Tukey Post-hoc test. adipocyte conversion of 3T3-L1 cells (Jessen and Stevens, RT-PCR 2002). Here, we have demonstrated that the Lcn2 transcript is Total RNA was extracted as above and 1-2 ␮g reverse-transcribed into cDNA using induced very quickly, from 6 hours after induction of adipose the avian myeloblastosis virus reverse transcriptase (Promega) and oligo-dT primer. ␣ PCR amplification was performed in 25 ␮l containing 1-2 ␮l of cDNA and 1.25 U differentiation, and that the absence of PKB or addition of a Taq DNA polymerase (Eppendorf). The oligonucleotides used in PCR analysis were PI 3-kinase inhibitor prevents increase of Lcn2 mRNA. Thus, as follows: Pparg, 5Ј-GGAAAGACAACGGACAAATCA-3Ј (sense) and 5Ј-ATC- it seems clear that Lcn2 is regulated through a PI 3-kinase/PKB CTTGGCCCTCTGAGATG-3Ј (antisense) (325 bp) (Croissandeau et al., 2002); aP2, 5Ј-GGAACCTGGAAGCTTGTCTCC-3Ј (sense) and 5Ј-ACCAGCTTGTCA- pathway during adipogenesis, but its precise role remains to be CCATCTCG-3Ј (antisense) (325 bp) (Hansen et al., 2001); ␤-actin, 5Ј-ATGG- determined. ATGACGATATCGCTGCGCTG-3Ј (sense) and 5Ј-CTAGAAGCACTTGCGGTG- In conclusion, we have demonstrated that PKB␣ is necessary CACGAT-3Ј (antisense) (1127 bp) (NM_007393); Klf15, 5Ј-CCCAATGCCGCCA- to trigger adipocyte differentiation of MEFs and have identified AACCTAT-3Ј (sense) and GAGGTGGCTGCTCTTGGTGTACATC (antisense) (161 bp) (Teshigawara et al., 2005); Ramp3, 5Ј-GGATGAAGTACTCATCCCAC- potential target genes affected by PKB␣ during the early phase TG-3Ј (sense) and 5Ј-GAATCGTGACAGATCACAGAG-3Ј (antisense) (665 bp) of adipogenesis. The mechanism of regulation of these (NM_019511); Ren1, 5Ј-CCTCACCAACTACCTGAATAC-3Ј (sense) and 5Ј-CA- candidates by PKB␣ and their contributions to the adipocyte CAGCCTTCTTCACATAGCA-3Ј (antisense) (622 bp) (NM_031192); Lcn2, 5Ј- CGATGTACAGCACCATCTATGAG-3Ј (sense) and 5Ј-CTCTCTGGCAACAGG- differentiation pathway await further investigation. AAAGATG-3Ј (antisense) (490 bp) (X14607). ␤-actin was used as an internal standard. PCR reaction mixtures were denatured at 94°C for 2 minutes and cDNA Materials and Methods templates amplified as followed: 20-30 cycles of denaturation at 94°C for 1 minute, annealing at 60-65°C for 1 minute, and extension at 72°C for 1 minute. At the end Cell culture and adipocyte differentiation of the cycling, the samples were incubated at 72°C for 7 minutes. The amplified Mouse embryonic fibroblasts (MEFs) were generated from 13.5-day-old embryos DNA products were visualised on 1-2% agarose gels and photographed under ␣ obtained from heterozygous PKB mice intercrosses (Yang et al., 2003). Briefly, ultraviolet light. Journal of Cell Science after dissection of head and visceral organs for genotyping, embryos were minced and trypsinised for 30 minutes at 37°C. Embryonic fibroblasts were then plated and Western blot analysis maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% foetal calf Cells were solubilised in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1% serum (FCS) (Life Technologies), 100 U/ml penicillin and 100 ␮g/ml streptomycin Nonidet P-40, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 2 ␮M at 37°C in an atmosphere of 5% CO . Genotype analysis was performed to 2 microcystin-LR (Alexis), 1 mM sodium pyrophosphate, 10 mM NaF and 0.1 mM determine cells carrying an homozygous null mutation for PKB␣ (Yang et al., 2003). sodium orthopervanadate. Lysates were centrifuged at 15,000 g for 10 minutes at All experiments were performed with wild-type and PKB␣–/– MEFs between 15-20 4°C and protein concentration determined using the Bradford reagent (Bio-Rad) with passages. 3T3-L1 preadipocytes were maintained in DMEM containing 10% FCS. BSA as standard. Protein extracts were separated by 7.5% or 10% SDS-PAGE and For adipocyte differentiation, 2-day-postconfluent cells (day 0) were treated with transferred onto Immobilon P membranes (Millipore) by electroblotting. After DMEM supplemented with 10% FCS, 8 ␮g/ml biotin, 4 ␮g/ml pantothenate, 0.5 blocking with 5% milk-TBST (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.1% mM 3-isobutyl-1-methylxanthine, 1 ␮M dexamethasone and 10 ␮g/ml insulin for Tween 20), membranes were incubated with rabbit polyclonal antibodies against 2 days (all from Sigma). Medium was renewed every 2 days with DMEM containing isoforms of PKB (Yang et al., 2003), phospho-PKB (Ser473 or Thr308) (Cell 10% FCS, 8 ␮g/ml biotin, 4 ␮g/ml pantothenate and 10 ␮g/ml insulin until cells Signaling Technologies) or with mouse monoclonal antibodies against pan-actin were used. LY294002 (Calbiochem) was prepared in dimethyl sulfoxide (DMSO) (NeoMarkers) or 12CA5 HA. Blots were then washed with TBST, incubated with and added at a final concentration of 50 ␮M in the medium 20 minutes before, and horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies every 12 hours, after addition of the adipose cocktail. To visualise lipid and detected using enhanced chemiluminescence reagents (Amersham Biosciences). accumulation, cells were stained with Oil Red O (Ramirez-Zacarias et al., 1992). Briefly, cells were washed with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde solution for 1 hour and stained with Oil Red O for 1 hour using a The authors thank E. Oakeley and H. Angliker for microarray 60:40 (v/v) dilution in water of a 0.5% stock solution (in isopropanol). Cells were analysis, and D. Hynx for help with the mice. The Friedrich Miescher then washed twice with PBS and twice with water. Institute for Biomedical Research is part of the Novartis Research Foundation. Retrovirus construction, production and infection The retroviral vectors expressing hemagglutinin-PKB␣ (HA-PKB␣) or (m/p)-HA- PKB␣ (constitutively active membrane-targeted form of PKB␣) were constructed by subcloning the BglII-EcoRI fragment from the pECE-HA-PKB␣ or pECE- References (m/p)-HA-PKB␣ vectors (Andjelkovic et al., 1997) into the pBABEpuro vector. Ailhaud, G., Teboul, M. and Massiera, F. (2002). Angiotensinogen, adipocyte differentiation and fat mass enlargement. Curr. Opin. Clin. Nutr. Metab. Care 5, 385-389. BOSC retrovirus packaging cells were cultured in DMEM with 10% FCS. To Alessi, D. R., Andjelkovic, M., Caudwell, F. B., Cron, P., Morrice, N., Cohen, P. and produce the retroviruses, BOSC cells were transiently transfected with 15 ␮g of ␣ ␣ Hemmings, B. A. (1996). Mechanism of activation of protein kinase B by insulin and the retroviral vector, HA-PKB or (m/p)-HA-PKB constructs by the calcium IGF-1. EMBO J. 15, 6541-6551. phosphate method. Forty-eight hours after transfection, viral supernatants were Andjelkovic, M., Alessi, D. R., Meier, R., Fernandez, A., Lamb, N. J., Frech, M., ␮ harvested, filtered through a 0.45 m membrane and applied to MEFs in 10 cm Cron, P., Cohen, P., Lucocq, J. M. and Hemmings, B. A. (1997). Role of dishes with 5 ␮g/ml polybrene (Sigma). A second infection of MEFs was translocation in the activation and function of protein kinase B. J. Biol. Chem. 272, performed after 8-12 hours. Twenty-four hours after infection with retrovirus, cells 31515-31524. PKB␣ and adipogenesis 897

Banerjee, S. S., Feinberg, M. W., Watanabe, M., Gray, S., Haspel, R. L., Denkinger, Mitogenic activation, phosphorylation and nuclear translocation of protein kinase B␤. D. J., Kawahara, R., Hauner, H. and Jain, M. K. (2003). The Kruppel-like factor J. Biol. Chem. 272, 30491-30497. KLF2 inhibits peroxisome proliferator-activated receptor-gamma expression and Miki, H., Yamauchi, T., Suzuki, R., Komeda, K., Tsuchida, A., Kubota, N., Terauchi, adipogenesis. J. Biol. Chem. 278, 2581-2584. Y., Kamon, J., Kaburagi, Y., Matsui, J. et al. (2001). Essential role of insulin receptor Brazil, D. P. and Hemmings, B. A. (2001). Ten years of protein kinase B signalling: a substrate 1 (IRS-1) and IRS-2 in adipocyte differentiation. Mol. Cell. Biol. 21, 2521- hard Akt to follow. Trends Biochem. Sci. 26, 657-664. 2532. Brodbeck, D., Cron, P. and Hemmings, B. A. (1999). A human protein kinase B␥ with Morfis, M., Christopoulos, A. and Sexton, P. M. (2003). RAMPs: 5 years on, where to regulatory phosphorylation sites in the activation loop and in the C-terminal now? Trends Pharmacol. Sci. 24, 596-601. hydrophobic domain. J. Biol. Chem. 274, 9133-9136. Mori, T., Sakaue, H., Iguchi, H., Gomi, H., Okada, Y., Takashima, Y., Nakamura, Chen, W. S., Xu, P. Z., Gottlob, K., Chen, M. L., Sokol, K., Shiyanova, T., Roninson, K., Nakamura, T., Yamauchi, T., Kubota, N. et al. (2005). Role of Kruppel-like I., Weng, W., Suzuki, R., Tobe, K. et al. (2001). Growth retardation and increased factor 15 (KLF15) in transcriptional regulation of adipogenesis. J. Biol. Chem. 280, apoptosis in mice with homozygous disruption of the Akt1 gene. Genes Dev. 15, 2203- 12867-12875. 2208. Nagae, T., Mukoyama, M., Sugawara, A., Mori, K., Yahata, K., Kasahara, M., Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, Suganami, T., Makino, H., Fujinaga, Y., Yoshioka, T. et al. (2000). Rat receptor- K. H., Bartolomei, M. S., Shulman, G. I. and Birnbaum, M. J. (2001a). Insulin activity-modifying proteins (RAMPs) for adrenomedullin/CGRP receptor: cloning and resistance and diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 upregulation in obstructive nephropathy. Biochem. Biophys. Res. Commun. 270, 89-93. (PKB beta). Science 292, 1728-1731. Nakae, J., Kitamura, T., Kitamura, Y., Biggs, W. H., Arden, K. and Accili, D. (2003). Cho, H., Thorvaldsen, J. L., Chu, Q., Feng, F. and Birnbaum, M. J. (2001b). The Forkhead Transcription Factor Foxo1 regulates adipocyte differentiation. Dev. Cell Akt1/PKB␣ is required for normal growth but dispensable for maintenance of glucose 4, 119-129. homeostasis in mice. J. Biol. Chem. 276, 38349-38352. Oishi, Y., Manabe, I., Tobe, K., Tsushima, K., Shindo, T., Fujiu, K., Nishimura, G., Christoffersen, C. T., Tornqvist, H., Vlahos, C. J., Bucchini, D., Jami, J., De Meyts, Maemura, K., Yamauchi, T., Kubota, N. et al. (2005). Krüppel-like transcription P. and Joshi, R. L. (1998). Insulin and insulin-like growth factor-I receptor mediated factor KLF5 is a key regulator of adipocyte differentiation. Cell Metab. 1, 27-39. differentiation of 3T3-F442A cells into adipocytes: effect of PI 3-kinase inhibition. Peng, X., Xu, P. Z., Chen, M. L., Hahn-Windgassen, A., Skeen, J., Jacobs, J., Biochem. Biophys. Res. Commun. 246, 426-430. Sundararajan, D., Chen, W. S., Crawford, S. E., Coleman, K. G. et al. (2003). Cinti, S., Eberbach, S., Castellucci, M. and Accili, D. (1998). Lack of insulin receptors Dwarfism, impaired skin development, atrophy, delayed bone affects the formation of white adipose tissue in mice. A morphometric and development, and impeded adipogenesis in mice lacking Akt 1 and Akt2. Genes Dev. ultrastructural analysis. Diabetologia 41, 171-177. 17, 1352-1365. Croissandeau, G., Basak, A., Seidah, N. G., Chretien, M. and Mbikay, M. (2002). Ramirez-Zacarias, J. L., Castro-Munozledo, F. and Kuri-Harcuch, W. (1992). Proprotein convertases are important mediators of the adipocyte differentiation of Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids mouse 3T3-L1 cells. J. Cell Sci. 115, 1203-1211. with Oil red O. Histochemistry 97, 493-497. Datta, S. R., Brunet, A. and Greenberg, M. E. (1999). Cellular survival: a play in three Rosen, E. D., Walkey, C. J., Puigserver, P. and Spiegelman, B. M. (2000). Akts. Genes Dev. 13, 2905-2927. Transcriptional regulation of adipogenesis. Genes Dev. 14, 1293-1307. Easton, R. M., Cho, H., Roovers, K., Shineman, D. W., Mizrahi, M., Forman, M. S., Saint-Marc, P., Kozak, L. P., Ailhaud, G., Darimont, C. and Negrel, R. (2001). Lee, V. M., Szabolcs, M., De Jong, R., Oltersdorf, T. et al. (2005). Role of Angiotensin II as a trophic factor of white adipose tissue: stimulation of adipose cell Akt3/protein kinase B␥ in attainment of normal brain size. Mol. Cell. Biol. 25, 1869- formation. Endocrinology 142, 487-492. 1878. Sakaue, H., Ogawa, W., Matsumoto, M., Kuroda, S., Takata, M., Sugimoto, T., Engeli, S., Negrel, R. and Sharma, A. M. (2000). Physiology and pathophysiology of Spiegelman, B. M. and Kasuga, M. (1998). Postranscriptional control of adipocyte the adipose tissue renin-angiotensin system. Hypertension 35, 1270-1277. differentiation through activation of phosphoinositide 3-kinase. J. Biol. Chem. 273, Flower, D. R. (1996). The lipocalin protein family: structure and function. Biochem. J. 28945-28952. 318, 1-14. Scheid, M. P. and Woodgett, J. R. (2001). PKB/AKT: functional insights from genetic Garofalo, R. S., Orena, S. J., Rafidi, K., Torchia, A. J., Stock, J. L., Hildebrandt, A. models. Nat. Rev. Mol. Cell. Biol. 2, 760-768. L., Coskran, T., Black, S. C., Brees, D. J., Wicks, J. R. et al. (2003). Severe diabetes, Schling, P. and Loffler, G. (2001). Effects of angiotensin II on adipose conversion and age-dependent loss of adipose tissue and mild growth deficiency in mice lacking expression of genes of the renin-angiotensin system in human preadipocytes. Horm. Akt2/PKB␤. J. Clin. Invest. 112, 197-208. Metab. Res. 33, 189-195. Gray, S., Feinberg, M. W., Hull, S., Kuo, C. T., Watanabe, M., Sen-Banerjee, S., Spiegelman, B. M., Choy, L., Hotamisligil, G. S., Graves, R. A. and Tontonoz, P. DePina, A., Haspel, R. and Jain, M. K. (2002). The Kruppel-like factor KLF15 (1993). Regulation of adipocyte gene expression in differentiation and syndromes of regulates the insulin-sensitive glucose transporter GLUT4. J. Biol. Chem. 277, 34322- /diabetes. J. Biol. Chem. 268, 6823-6826. 34328. Smith, P. J., Wise, L. S., Berkowitz, R., Wan, C. and Rubin, C. S. (1988). Insulin-like Journal of Cell Science Green, H. and Meuth, M. (1974). An established preadipose cell line and its growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes. J. differentiation in culture. Cell 3, 127-133. Biol. Chem. 263, 9402-9408. Gregoire, F. M., Smas, C. M. and Sul, H. S. (1998). Understanding adipocyte Teshigawara, K., Ogawa, W., Mori, T., Matsuki, Y., Watanabe, E., Hiramatsu, R., differentiation. Physiol. Rev. 78, 783-809. Inoue, H., Miyake, K., Sakaue, H. and Kasuga, M. (2005). Role of Kruppel-like Hansen, J. B., Zhang, H., Rasmussen, T. H., Petersen, R. K., Flindt, E. N. and factor 15 in PEPCK gene expression in the . Biochem. Biophys. Res. Commun. Kristiansen, K. (2001). Peroxisome proliferator-activated receptor delta (PPARdelta)- 327, 920-926. mediated regulation of preadipocyte proliferation and gene expression is dependent on Tomiyama, K., Nakata, H., Sasa, H., Arimura, S., Nishio, E. and Watanabe, Y. cAMP signaling. J. Biol. Chem. 276, 3175-3182. (1995). Wortmannin, a specific phosphatidylinositol 3-kinase inhibitor, inhibits Hill, M. M. and Hemmings, B. A. (2002). Inhibition of protein kinase B/Akt: adipocytic differentiation of 3T3-L1 cells. Biochem. Biophys. Res. Commun. 212, 263- implications for cancer therapy. Pharmacol. Ther. 93, 243-251. 269. Jessen, B. A. and Stevens, G. J. (2002). Expression profiling during adipocyte Tschopp, O., Yang, Z. Z., Brodbeck, D., Hemmings-Mieszczak, M., Dummler, B., differentiation of 3T3-L1 fibroblasts. Gene 299, 95-100. Watanabe, T., Michaelis, T., Frahm, J. and Hemmings, B. A. (2005). Reduced brain Kohn, A. D., Summers, S. A., Birnbaum, M. J. and Roth, R. A. (1996). Expression of size in protein kinase B␥ (PKB␥/Akt3) null mutant mice. Development 132, 2943- a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose 2954. uptake and glucose transporter 4 translocation. J. Biol. Chem. 271, 31372-31378. Whiteman, E. L., Cho, H. and Birnbaum, M. J. (2002). Role of Akt/protein kinase B Kratchmarova, I., Kalume, D. E., Blagoev, B., Scherer, P. E., Podtelejnikov, A. V., in metabolism. Trends Endocrinol. Metab. 13, 444-451. Molina, H., Bickel, P. E., Andersen, J. S., Fernandez, M. M., Bunkenborg, J. et al. Xia, X. and Serrero, G. (1999). Inhibition of adipose differentiation by (2002). A proteomic approach for identification of secreted proteins during the phosphatidylinositol 3-kinase inhibitors. J. Cell. Physiol. 178, 9-16. differentiation of 3T3-L1 preadipocytes to adipocytes. Mol. Cell Proteomics 1, 213- Xu, J. and Liao, K. (2004). Protein kinase B/AKT 1 plays a pivotal role in insulin-like 222. growth factor-1 receptor signaling induced 3T3-L1 adipocyte differentiation. J. Biol. Lawlor, M. A. and Alessi, D. R. (2001). PKB/Akt: a key mediator of cell proliferation, Chem. 279, 35914-35922. survival and insulin responses? J. Cell Sci. 114, 2903-2910. Yang, Z. Z., Tschopp, O., Hemmings-Mieszczak, M., Feng, J., Brodbeck, D., Li, Y., Totsune, K., Takeda, K., Furuyama, K., Shibahara, S. and Takahashi, K. Perentes, E. and Hemmings, B. A. (2003). Protein kinase B␣/Akt1 regulates placental (2003). Differential expression of adrenomedullin and resistin in 3T3-L1 adipocytes development and fetal growth. J. Biol. Chem. 278, 32124-32131. treated with tumor necrosis factor-alpha. Eur. J. Endocrinol. 149, 231-238. Yang, Z. Z., Tschopp, O., Baudry, A., Dummler, B., Hynx, D. and Hemmings, B. A. Magun, R., Burgering, B. M., Coffer, P. J., Pardasani, D., Lin, Y., Chabot, J. and (2004). Physiological functions of protein kinase B/Akt. Biochem. Soc. Trans. 32, 350- Sorisky, A. (1996). Expression of a constitutively activated form of protein kinase B 354. (c-Akt) in 3T3-L1 preadipose cells causes spontaneous differentiation. Endocrinology Yeh, W. C., Cao, Z., Classon, M. and McKnight, S. L. (1995). Cascade regulation of 137, 3590-3593. terminal adipocyte differentiation by three members of the C/EBP family of leucine Meier, R., Alessi, D. R., Cron, P., Andjelkovic, M. and Hemmings, B. A. (1997). zipper proteins. Genes Dev. 9, 168-181.